Nuclear chromosome locations dictate segregation error frequencies.

Chromosome segregation errors during cell divisions generate aneuploidies and micronuclei, which can undergo extensive chromosomal rearrangements such as chromothripsis1-5. Selective pressures then shape distinct aneuploidy and rearrangement patterns-for example, in cancer6,7-but it is unknown whether initial biases in segregation errors and micronucleation exist for particular chromosomes. Using single-cell DNA sequencing8 after an error-prone mitosis in untransformed, diploid cell lines and organoids, we show that chromosomes have different segregation error frequencies that result in non-random aneuploidy landscapes. Isolation and sequencing of single micronuclei from these cells showed that mis-segregating chromosomes frequently also preferentially become entrapped in micronuclei. A similar bias was found in naturally occurring micronuclei of two cancer cell lines. We find that segregation error frequencies of individual chromosomes correlate with their location in the interphase nucleus, and show that this is highest for peripheral chromosomes behind spindle poles. Randomization of chromosome positions, Cas9-mediated live tracking and forced repositioning of individual chromosomes showed that a greater distance from the nuclear centre directly increases the propensity to mis-segregate. Accordingly, chromothripsis in cancer genomes9 and aneuploidies in early development10 occur more frequently for larger chromosomes, which are preferentially located near the nuclear periphery. Our findings reveal a direct link between nuclear chromosome positions, segregation error frequencies and micronucleus content, with implications for our understanding of tumour genome evolution and the origins of specific aneuploidies during development.

Etiological involvement of KCND1 variants in an X-linked neurodevelopmental disorder with variable expressivity.

Utilizing trio whole-exome sequencing and a gene matching approach, we identified a cohort of 18 male individuals from 17 families with hemizygous variants in KCND1, including two de novo missense variants, three maternally inherited protein-truncating variants, and 12 maternally inherited missense variants. Affected subjects present with a neurodevelopmental disorder characterized by diverse neurological abnormalities, mostly delays in different developmental domains, but also distinct neuropsychiatric signs and epilepsy. Heterozygous carrier mothers are clinically unaffected. KCND1 encodes the α-subunit of Kv4.1 voltage-gated potassium channels. All variant-associated amino acid substitutions affect either the cytoplasmic N- or C-terminus of the channel protein except for two occurring in transmembrane segments 1 and 4. Kv4.1 channels were functionally characterized in the absence and presence of auxiliary ß subunits. Variant-specific alterations of biophysical channel properties were diverse and varied in magnitude. Genetic data analysis in combination with our functional assessment shows that Kv4.1 channel dysfunction is involved in the pathogenesis of an X-linked neurodevelopmental disorder frequently associated with a variable neuropsychiatric clinical phenotype.

The clinical and molecular spectrum of the KDM6B-related neurodevelopmental disorder.

De novo variants are a leading cause of neurodevelopmental disorders (NDDs), but because every monogenic NDD is different and usually extremely rare, it remains a major challenge to understand the complete phenotype and genotype spectrum of any morbid gene. According to OMIM, heterozygous variants in KDM6B cause "neurodevelopmental disorder with coarse facies and mild distal skeletal abnormalities." Here, by examining the molecular and clinical spectrum of 85 reported individuals with mostly de novo (likely) pathogenic KDM6B variants, we demonstrate that this description is inaccurate and potentially misleading. Cognitive deficits are seen consistently in all individuals, but the overall phenotype is highly variable. Notably, coarse facies and distal skeletal anomalies, as defined by OMIM, are rare in this expanded cohort while other features are unexpectedly common (e.g., hypotonia, psychosis, etc.). Using 3D protein structure analysis and an innovative dual Drosophila gain-of-function assay, we demonstrated a disruptive effect of 11 missense/in-frame indels located in or near the enzymatic JmJC or Zn-containing domain of KDM6B. Consistent with the role of KDM6B in human cognition, we demonstrated a role for the Drosophila KDM6B ortholog in memory and behavior. Taken together, we accurately define the broad clinical spectrum of the KDM6B-related NDD, introduce an innovative functional testing paradigm for the assessment of KDM6B variants, and demonstrate a conserved role for KDM6B in cognition and behavior. Our study demonstrates the critical importance of international collaboration, sharing of clinical data, and rigorous functional analysis of genetic variants to ensure correct disease diagnosis for rare disorders.

Rare germline heterozygous missense variants in BRCA1-associated protein 1, BAP1, cause a syndromic neurodevelopmental disorder.

Nuclear deubiquitinase BAP1 (BRCA1-associated protein 1) is a core component of multiprotein complexes that promote transcription by reversing the ubiquitination of histone 2A (H2A). BAP1 is a tumor suppressor whose germline loss-of-function variants predispose to cancer. To our knowledge, there are very rare examples of different germline variants in the same gene causing either a neurodevelopmental disorder (NDD) or a tumor predisposition syndrome. Here, we report a series of 11 de novo germline heterozygous missense BAP1 variants associated with a rare syndromic NDD. Functional analysis showed that most of the variants cannot rescue the consequences of BAP1 inactivation, suggesting a loss-of-function mechanism. In T cells isolated from two affected children, H2A deubiquitination was impaired. In matching peripheral blood mononuclear cells, histone H3 K27 acetylation ChIP-seq indicated that these BAP1 variants induced genome-wide chromatin state alterations, with enrichment for regulatory regions surrounding genes of the ubiquitin-proteasome system (UPS). Altogether, these results define a clinical syndrome caused by rare germline missense BAP1 variants that alter chromatin remodeling through abnormal histone ubiquitination and lead to transcriptional dysregulation of developmental genes.

CAPRIN1 haploinsufficiency causes a neurodevelopmental disorder with language impairment, ADHD and ASD.

We describe an autosomal dominant disorder associated with loss-of-function variants in the Cell cycle associated protein 1 (CAPRIN1; MIM*601178). CAPRIN1 encodes a ubiquitous protein that regulates the transport and translation of neuronal mRNAs critical for synaptic plasticity, as well as mRNAs encoding proteins important for cell proliferation and migration in multiple cell types. We identified 12 cases with loss-of-function CAPRIN1 variants, and a neurodevelopmental phenotype characterized by language impairment/speech delay (100%), intellectual disability (83%), attention deficit hyperactivity disorder (82%) and autism spectrum disorder (67%). Affected individuals also had respiratory problems (50%), limb/skeletal anomalies (50%), developmental delay (42%) feeding difficulties (33%), seizures (33%) and ophthalmologic problems (33%). In patient-derived lymphoblasts and fibroblasts, we showed a monoallelic expression of the wild-type allele, and a reduction of the transcript and protein compatible with a half dose. To further study pathogenic mechanisms, we generated sCAPRIN1+/- human induced pluripotent stem cells via CRISPR-Cas9 mutagenesis and differentiated them into neuronal progenitor cells and cortical neurons. CAPRIN1 loss caused reduced neuronal processes, overall disruption of the neuronal organization and an increased neuronal degeneration. We also observed an alteration of mRNA translation in CAPRIN1+/- neurons, compatible with its suggested function as translational inhibitor. CAPRIN1+/- neurons also showed an impaired calcium signalling and increased oxidative stress, two mechanisms that may directly affect neuronal networks development, maintenance and function. According to what was previously observed in the mouse model, measurements of activity in CAPRIN1+/- neurons via micro-electrode arrays indicated lower spike rates and bursts, with an overall reduced activity. In conclusion, we demonstrate that CAPRIN1 haploinsufficiency causes a novel autosomal dominant neurodevelopmental disorder and identify morphological and functional alterations associated with this disorder in human neuronal models.

Clinical diversity and molecular mechanism of VPS35L-associated Ritscher-Schinzel syndrome.

PURPOSE: The Retriever subunit VPS35L is the third responsible gene for Ritscher-Schinzel syndrome (RSS) after WASHC5 and CCDC22. To date, only one pair of siblings have been reported and their condition was significantly more severe than typical RSS. This study aimed to understand the clinical spectrum and underlying molecular mechanism in VPS35L-associated RSS. METHODS: We report three new patients with biallelic VPS35L variants. Biochemical and cellular analyses were performed to elucidate disease aetiology. RESULTS: In addition to typical features of RSS, we confirmed hypercholesterolaemia, hypogammaglobulinaemia and intestinal lymphangiectasia as novel complications of VPS35L-associated RSS. The latter two complications as well as proteinuria have not been reported in patients with CCDC22 and WASHC5 variants. One patient showed a severe phenotype and the other two were milder. Cells established from patients with the milder phenotypes showed relatively higher VPS35L protein expression. Cellular analysis found VPS35L ablation decreased the cell surface level of lipoprotein receptor-related protein 1 and low-density lipoprotein receptor, resulting in reduced low-density lipoprotein cellular uptake. CONCLUSION: VPS35L-associated RSS is a distinct clinical entity with diverse phenotype and severity, with a possible molecular mechanism of hypercholesterolaemia. These findings provide new insight into the essential and distinctive role of Retriever in human development.

Delineation of a KDM2B-related neurodevelopmental disorder and its associated DNA methylation signature.

PURPOSE: Pathogenic variants in genes involved in the epigenetic machinery are an emerging cause of neurodevelopment disorders (NDDs). Lysine-demethylase 2B (KDM2B) encodes an epigenetic regulator and mouse models suggest an important role during development. We set out to determine whether KDM2B variants are associated with NDD. METHODS: Through international collaborations, we collected data on individuals with heterozygous KDM2B variants. We applied methylation arrays on peripheral blood DNA samples to determine a KDM2B associated epigenetic signature. RESULTS: We recruited a total of 27 individuals with heterozygous variants in KDM2B. We present evidence, including a shared epigenetic signature, to support a pathogenic classification of 15 KDM2B variants and identify the CxxC domain as a mutational hotspot. Both loss-of-function and CxxC-domain missense variants present with a specific subepisignature. Moreover, the KDM2B episignature was identified in the context of a dual molecular diagnosis in multiple individuals. Our efforts resulted in a cohort of 21 individuals with heterozygous (likely) pathogenic variants. Individuals in this cohort present with developmental delay and/or intellectual disability; autism; attention deficit disorder/attention deficit hyperactivity disorder; congenital organ anomalies mainly of the heart, eyes, and urogenital system; and subtle facial dysmorphism. CONCLUSION: Pathogenic heterozygous variants in KDM2B are associated with NDD and a specific epigenetic signature detectable in peripheral blood.

De novo putative loss-of-function variants in TAF4 are associated with a neuro-developmental disorder.

TATA-binding protein associated factor 4 (TAF4) is a subunit of the Transcription Factor IID (TFIID) complex, a central player in transcription initiation. Other members of this multimeric complex have been implicated previously as monogenic disease genes in human developmental disorders. TAF4 has not been described to date as a monogenic disease gene. We here present a cohort of eight individuals, each carrying de novo putative loss-of-function (pLoF) variants in TAF4 and expressing phenotypes consistent with a neuro-developmental disorder (NDD). Common features include intellectual disability, abnormal behavior, and facial dysmorphisms. We propose TAF4 as a novel dominant disease gene for NDD, and coin this novel disorder "TAF4-related NDD" (T4NDD). We place T4NDD in the context of other disorders related to TFIID subunits, revealing shared features of T4NDD with other TAF-opathies.

Variants in CAPZA2, a member of an F-actin capping complex, cause intellectual disability and developmental delay.

The actin cytoskeleton is regulated by many proteins including capping proteins that stabilize actin filaments (F-actin) by inhibiting actin polymerization and depolymerization. Here, we report two pediatric probands who carry damaging heterozygous de novo mutations in CAPZA2 (HGNC: 1490) and exhibit neurological symptoms with shared phenotypes including global motor development delay, speech delay, intellectual disability, hypotonia and a history of seizures. CAPZA2 encodes a subunit of an F-actin-capping protein complex (CapZ). CapZ is an obligate heterodimer consisting of α and ß heterodimer conserved from yeast to human. Vertebrate genomes contain three α subunits encoded by three different genes and CAPZA2 encodes the α2 subunit. The single orthologue of CAPZA genes in Drosophila is cpa. Loss of cpa leads to lethality in early development and expression of the human reference; CAPZA2 rescues this lethality. However, the two CAPZA2 variants identified in the probands rescue this lethality at lower efficiency than the reference. Moreover, expression of the CAPZA2 variants affects bristle morphogenesis, a process that requires extensive actin polymerization and bundling during development. Taken together, our findings suggest that variants in CAPZA2 lead to a non-syndromic neurodevelopmental disorder in children.

A gene-to-patient approach uplifts novel disease gene discovery and identifies 18 putative novel disease genes.

PURPOSE: Exome and genome sequencing have drastically accelerated novel disease gene discoveries. However, discovery is still hindered by myriad variants of uncertain significance found in genes of undetermined biological function. This necessitates intensive functional experiments on genes of equal predicted causality, leading to a major bottleneck. METHODS: We apply the loss-of-function observed/expected upper-bound fraction metric of intolerance to gene inactivation to curate a list of predicted haploinsufficient disease genes. Using data from the 100,000 Genomes Project, we adopt a gene-to-patient approach that matches de novo loss-of-function variants in constrained genes to patients with rare disease. Through large-scale aggregation of data, we reduce excess analytical noise currently hindering novel discoveries. RESULTS: Results from 13,949 trios revealed 643 rare, de novo predicted loss-of-function events filtered from 1044 loss-of-function observed/expected upper-bound fraction-constrained genes. A total of 168 variants occurred within 126 genes without a known disease-gene relationship. Of these, 27 genes had >1 kindred affected, and for 18 of these genes, multiple kindreds had overlapping phenotypes. Two years after initial analysis, 11 of 18 (61%) of these genes have been independently published as novel disease gene discoveries. CONCLUSION: Using large cohorts and adopting gene-based approaches can rapidly and objectively accelerate dominantly inherited novel gene discovery by targeting the most appropriate genes for functional validation.