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PCDH19-Clustering Epilepsy (PCDH19-CE) is an infantile onset disorder caused by mutation of the X-linked PCDH19 gene. Intriguingly, heterozygous females are affected while hemizygous males are not. While there is compelling evidence that this disorder stems from the coexistence of WT and PCDH19-null cells, the cellular mechanism underpinning the neurological phenotype remains unclear. Here, we investigate the impact of Pcdh19 WT and KO neuron mosaicism on synaptogenesis and network activity. Using our previously established knock-in and knock-out mouse models, together with CRISPR-Cas9 genome editing technology, we demonstrate a reduction in excitatory synaptic contacts between PCDH19-expressing and PCDH19-null neurons. Significantly altered neuronal morphology and neuronal network activities were also identified in the mixed populations. In addition, we show that in Pcdh19 heterozygous mice, where the coexistence of WT and KO neurons naturally occurs, aberrant contralateral axonal branching is present. Overall, our data show that mosaic expression of PCDH19 disrupts physiological neurite communication leading to abnormal neuronal activity, a hallmark of PCDH19-CE. Refereed/Peer-reviewed

Currently, there are at least a dozen recognized hereditary hematopoietic malignancies (HHMs), some of which phenocopy others. Among these, three HHMs driven by germline mutations in ANKRD26, ETV6, or RUNX1 share a phenotype of thrombocytopenia, qualitative platelet defects, and an increased lifetime risk of hematopoietic malignancies (HMs). Prior work has demonstrated that RUNX1 germline mutation carriers experience an elevated lifetime risk (66%) for developing clonal hematopoiesis (CH) prior to age 50. Germline mutations in ANKRD26 or ETV6 phenocopy RUNX1 germline mutations, but no studies have focused on the risk of CH in individuals with germline mutations in ANKRD26 or ETV6.To determine the prevalence of CH in individuals with germline mutations in ANKRD26 or ETV6, we performed next generation sequencing on hematopoietic tissue from twelve individuals with either germline ANKRD26 or germline ETV6 mutations. Each patient had thrombocytopenia but had not developed HMs. Among the seven individuals with germline ANKRD26 mutations, one patient had a CH clone driven by a somatic SF3B1 mutation (p.Lys700Glu). This mutation increased from a variant allele frequency (VAF) of 9.4% at age 56 to 17.4% at age 60. None of the germline ETV6 mutation carriers had evidence of CH at the limits of detection of the NGS assay (5% VAF). Unlike individuals with germline mutations in RUNX1, no individuals under the age of 50 with germline mutations in ANKRD26 or ETV6 had detectable CH. This work demonstrates that ANKRD26 germline mutation carriers, but not ETV6 mutation carriers, experience elevated risk for CH.

Refereed/Peer-reviewed GATA2 deficiency syndrome (G2DS) is a rare autosomal dominant genetic disease predisposing to a range of symptoms, of which myeloid malignancy and immunodeficiency including recurrent infections are most common. In the last decade since it was first reported, there have been over 480 individuals identified carrying a pathogenic or likely pathogenic germline GATA2 variant with symptoms of G2DS, with 240 of these confirmed to be familial and 24 de novo. For those that develop myeloid malignancy (75% of all carriers with G2DS disease symptoms), the median age of onset is 17 years (range 0-78 years) and myelodysplastic syndrome is the first diagnosis in 75% of these cases with acute myeloid leukemia in a further 9%. All variant types appear to predispose to myeloid malignancy and immunodeficiency. Apart from lymphedema in which haploinsufficiency seems necessary, the mutational requirements of the other less common G2DS phenotypes is still unclear. These predominantly loss-of-function variants impact GATA2 expression and function in numerous ways including perturbations to DNA binding, protein structure, protein:protein interactions, and gene transcription, splicing, and expression. In this review, we provide the first expert-curated ACMG/AMP classification with codes of published variants compatible for use in clinical or diagnostic settings.

PCDH19-Girls Clustering Epilepsy (PCDH19-GCE) is a childhood epileptic encephalopathy characterised by a spectrum of neurodevelopmental problems. PCDH19-GCE is caused by heterozygous loss-of-function mutations in the X-chromosome gene, Protocadherin 19 (PCDH19) encoding a cell-cell adhesion molecule. Intriguingly, hemizygous males are generally unaffected. As PCDH19 is subjected to random X-inactivation, heterozygous females are comprised of a mosaic of cells expressing either the normal or mutant allele, which is thought to drive pathology. Despite being the second most prevalent monogeneic cause of epilepsy, little is known about the role of PCDH19 in brain development. In this study we show that PCDH19 is highly expressed in human neural stem and progenitor cells (NSPCs) and investigate its function in vitro in these cells of both mouse and human origin. Transcriptomic analysis of mouse NSPCs lacking Pcdh19 revealed changes to genes involved in regulation of neuronal differentiation, and we subsequently show that loss of Pcdh19 causes increased NSPC neurogenesis. We reprogramed human fibroblast cells harbouring a pathogenic PCDH19 mutation into human induced pluripotent stem cells (hiPSC) and employed neural differentiation of these to extend our studies into human NSPCs. As in mouse, loss of PCDH19 function caused increased neurogenesis, and furthermore, we show this is associated with a loss of human NSPC polarity. Overall our data suggests a conserved role for PCDH19 in regulating mammalian cortical neurogenesis and has implications for the pathogenesis of PCDH19-GCE. We propose that the difference in timing or “heterochrony” of neuronal cell production originating from PCDH19 wildtype and mutant NSPCs within the same individual may lead to downstream asynchronies and abnormalities in neuronal network formation, which in-part predispose the individual to network dysfunction and epileptic activity. Refereed/Peer-reviewed

First reported in 1999, germline runt-related transcription factor 1 (RUNX1) mutations are a well-established cause of familial platelet disorder with predisposition to myeloid malignancy (FPD-MM). We present the clinical phenotypes and genetic mutations detected in 10 novel RUNX1-mutated FPD-MM families. Genomic analyses on these families detected 2 partial gene deletions, 3 novel mutations, and 5 recurrent mutations as the germline RUNX1 alterations leading to FPD-MM. Combining genomic data from the families reported herein with aggregated published data sets resulted in 130 germline RUNX1 families, which allowed us to investigate whether specific germline mutation characteristics (type, location) could explain the large phenotypic heterogeneity between patients with familial platelet disorder and different HMs. Comparing the somatic mutational signatures between the available familial (n = 35) and published sporadic (n = 137) RUNX1-mutated AML patients showed enrichment for somatic mutations affecting the second RUNX1 allele and GATA2. Conversely, we observed a decreased number of somatic mutations affecting NRAS, SRSF2, and DNMT3A and the collective genes associated with CHIP and epigenetic regulation. This is the largest aggregation and analysis of germline RUNX1 mutations performed to date, providing a unique opportunity to examine the factors underlying phenotypic differences and disease progression from FPD to MM.

We show that the loss of Iqsec2 function in mice recapitulates key aspects of the human phenotype, irrespective of the X-inactivation status of the gene between species. Our understanding of the traditional X-chromosome inheritance with heterozygous female sparing needs revisiting., Clinical presentations of mutations in the IQSEC2 gene on the X-chromosome initially implicated to cause non-syndromic intellectual disability (ID) in males have expanded to include early onset seizures in males as well as in females. The molecular pathogenesis is not well understood, nor the mechanisms driving disease expression in heterozygous females. Using a CRISPR/Cas9–edited Iqsec2 KO mouse model, we confirm the loss of Iqsec2 mRNA expression and lack of Iqsec2 protein within the brain of both founder and progeny mice. Both male (52%) and female (46%) Iqsec2 KO mice present with frequent and recurrent seizures. Focusing on Iqsec2 KO heterozygous female mice, we demonstrate increased hyperactivity, altered anxiety and fear responses, decreased social interactions, delayed learning capacity and decreased memory retention/novel recognition, recapitulating psychiatric issues, autistic-like features, and cognitive deficits present in female patients with loss-of-function IQSEC2 variants. Despite Iqsec2 normally acting to activate Arf6 substrate, we demonstrate that mice modelling the loss of Iqsec2 function present with increased levels of activated Arf6. We contend that loss of Iqsec2 function leads to altered regulation of activated Arf6-mediated responses to synaptic signalling and immature synaptic networks. We highlight the importance of IQSEC2 function for females by reporting a novel nonsense variant c.566C > A, p.(S189*) in an elderly female patient with profound intellectual disability, generalised seizures, and behavioural disturbances. Our human and mouse data reaffirm IQSEC2 as another disease gene with an unexpected X-chromosome heterozygous female phenotype. Our Iqsec2 mouse model recapitulates the phenotypes observed in human patients despite the differences in the IQSEC2/Iqsec2 gene X-chromosome inactivation between the species.

Highly conserved TREX-mediated mRNA export is emerging as a key pathway in neuronal development and differentiation. TREX subunit variants cause neurodevelopmental disorders (NDDs) by interfering with mRNA export from the cell nucleus to the cytoplasm. Previously we implicated four missense variants in the X-linked THOC2 gene in intellectual disability (ID). We now report an additional six affected individuals from five unrelated families with two de novo and three maternally inherited pathogenic or likely pathogenic variants in THOC2 extending the genotypic and phenotypic spectrum. These comprise three rare missense THOC2 variants that affect evolutionarily conserved amino acid residues and reduce protein stability and two with canonical splice-site THOC2 variants that result in C-terminally truncated THOC2 proteins. We present detailed clinical assessment and functional studies on a de novo variant in a female with an epileptic encephalopathy and discuss an additional four families with rare variants in THOC2 with supportive evidence for pathogenicity. Severe neurocognitive features, including movement and seizure disorders, were observed in this cohort. Taken together our data show that even subtle alterations to the canonical molecular pathways such as mRNA export, otherwise essential for cellular life, can be compatible with life, but lead to NDDs in humans. Refereed/Peer-reviewed

DEAD-Box helicase 41 (DDX41) is one of the most commonly reported familial hematological malignancy (HM) genes, first reported in 2015. Mutated, it predisposes to both MDS/AML and lymphoma with an age of diagnosis similar to that of sporadic cases. Consequently, unrecognized DDX41 mutated cases are present in 'sporadic' cohorts with families often identified this way. Individuals with DDX41 mutation or deficiency have generally poor outcome with no effective targeted therapies available. The biological mechanism by which mutant DDX41 predisposes to HM is poorly understood making risk assessment for asymptomatic carriers difficult. In over 50% of HM affected individuals with germline DDX41 mutations, an acquired somatic mutation is identified on the other DDX41 allele with R525H being most common. Analysis of other somatic mutations in germline DDX41 mutation carriers reveals a minimal fingerprint of acquired somatic alterations in known myeloid malignancy driver genes with an absence of NPM1 and FLT3 mutations and an under-representation of mutations in DNMT3A, TET2, IDH1/2 and RUNX1. This may indicate a mechanism of leukemia development unique to germline DDX41 cases. Through an integrative combinatorial genomics approach of whole exome sequencing, leukemic panel sequencing and RNA-sequencing, we are in the process of comprehensively assessing six independent DDX41 germline mutated families. We and others have identified that DDX41 mutations are recurrently associated with MDS with erythroid dysplasia and erythroid subtype leukemia consistent with functional in vitro experiments showing that DDX41 mutations including loss-of-function can impact erythroid differentiation. Our preliminary RNA-Seq analysis on peripheral blood mononuclear cells of DDX41 carriers with HM reveals altered expression of genes involved in hemoglobin complex and innate immunity. DDX41 is reported to bind RNA splicing components such as SF3B1 and PRPF8 which are recurrently mutated in MDS/AML and intriguingly, the R525H mutation disrupts this interaction. Further analysis of our RNA-Seq data interrogating alternative splicing, gene expression analysis and biological pathways to uncover mechanisms for mutant DDX41 oncogenicity is currently ongoing. We have used CRISPR-Cas9 technology to generate Ddx41M1I mice as a model for one of the most common germline mutations in familial HM to investigate the direct effect of DDX41 mutation on hematopoiesis. Preliminary results indicate a sub-clinical reduction in hemoglobin levels in mice carrying the M1I mutation in heterozygosity when compared to their wildtype littermates. We are further investigating these mice with particular emphasis on characterizing hematopoiesis longitudinally to better understand mechanisms of disease onset and progression. This integrative genomic and functional approach to evaluate both mutations and biological pathways affected in DDX41 mutated malignancies will provide biological insight facilitating advancement in diagnosis, risk assessment, monitoring and personalized treatment. Disclosures Scott: Celgene: Honoraria.

DEAD-Box helicase 41 (DDX41) is the most commonly reported familial haematological malignancy (HM) gene first reported in 2015. Mutated, it predisposes to both MDS/AML and lymphoma, with HM diagnosis age like that of sporadic malignancies. Individuals with DDX41 mutation or deficiency have generally poor outcome with no effective targeted therapies available. Biological function of DDX41 leading to HM predisposition is poorly understood making risk assessment for asymptomatic carriers difficult. In over 50% of reported affected DDX41 germline mutated individuals, acquired somatic mutations are commonly identified on the other DDX41 allele. Through an integrative combinatorial genomics approach of whole exome sequencing, leukemic panel sequencing and RNA-sequencing, we comprehensively assessed six independent DDX41 germline mutated families. Each family presented varying penetrance of HM with affected and unaffected carriers as well as wildtype individuals assessed. Blood and bone marrow samples were assessed for the identification of somatic alterations in leukaemia associated genes in both malignant and pre-leukemic states. Interestingly, in both affected and unaffected DDX41 germline mutation carriers a minimal fingerprint of acquired somatic alterations was observed, potentially indicating a mechanism of leukaemia development unique to DDX41 mutations. Analysis of RNA-seq data through splicing and gene expression analysis to elucidate biological pathways which may be altered in DDX41 malignancy progression is ongoing. This integrative genomic approach to evaluate both mutations and biological pathways affected in DDX41 mutated malignancies will provide biological insight facilitating an advancement in diagnosis, risk assessment, monitoring and personalised treatment.

Over 100 different families have been reported with damaging germline mutations or deletions in RUNX1, making it a clinically significant gene in FPD-MM. In large families, the diagnosis of haematological malignancy (HM) shows variable penetrance among members with the same mutation; some carriers do not develop HM. The cause of this heterogeneity is not yet understood but it is likely attributable to additional somatic genetic changes in these germline RUNX1 carriers. To better understand the genetic landscape surrounding RUNX1 germline predisposition to HM we have developed an interactive online database, the RUNX1 database ( https://runx1db.runx1.com ). This database is designed for the sharing and curation of genetic data acquired from individuals with germline RUNX1 mutation both pre- and post-HM progression. Currently we have collected 170 datasets from 11 countries. Raw NGS data files were transferred, preferentially as FASTQ files, and run through a standardised bioinformatics pipeline generating VCF and BAM files. These files were uploaded onto the RUNX1.db where the integrated VariantGrid, genomics analysis software, can be used to provide automated annotation of variants within the VCF files. Using the RUNX1db to curate variants from previously published and new datasets we have been able to confirm published somatic and germline mutations, and importantly, extend on these findings by identifying additional somatic mutations of clinical significance. Interrogating this dataset as a single cohort we have identified somatic genetic signatures in pre-leukemic samples prior progression to AML, including recurrent mutations in several clonal haematopoiesis genes including BCOR. This RUNX1 worldwide genomics cohort will lead to powerful insights in the development of precision-based medicine diagnosis, risk assessment, monitoring and therapeutic intervention.

With a wealth of disease-associated DNA variants being recently reported, the challenges of providing their functional characterization are mounting. Previously, as part of a large systematic resequencing of the X chromosome in 208 unrelated families with nonsyndromic X-linked intellectual disability, we identified three unique variants (two missense and one protein truncating) in USP9X. To assess the functional significance of these variants, we took advantage of the Usp9x knockout mouse we generated. Loss of Usp9x causes reduction in both axonal growth and neuronal cell migration. Although overexpression of wild-type human USP9X rescued these defects, all three USP9X variants failed to rescue axonal growth, caused reduced USP9X protein localization in axonal growth cones, and (in 2/3 variants) failed to rescue neuronal cell migration. Interestingly, in one of these families, the proband was subsequently identified to have a microdeletion encompassing ARID1B, a known ID gene. Given our findings it is plausible that loss of function of both genes contributes to the individual's phenotype. This case highlights the complexity of the interpretations of genetic findings from genome-wide investigations. We also performed proteomics analysis of neurons from both the wild-type and Usp9x knockout embryos and identified disruption of the cytoskeleton as the main underlying consequence of the loss of Usp9x. Detailed clinical assessment of all three families with USP9X variants identified hypotonia and behavioral and morphological defects as common features in addition to ID. Together our data support involvement of all three USP9X variants in ID in these families and provide likely cellular and molecular mechanisms involved. Refereed/Peer-reviewed

Protocadherin 19 (Pcdh19) is an X-linked gene belonging to the protocadherin superfamily, whose members are predominantly expressed in the central nervous system and have been implicated in cell-cell adhesion, axon guidance and dendrite self-avoidance. Heterozygous loss-of-function mutations in humans result in the childhood epilepsy disorder PCDH19 Girls Clustering Epilepsy (PCDH19 GCE) indicating that PCDH19 is required for brain development. However, understanding PCDH19 function in vivo has proven challenging and has not been studied in mammalian models. Here, we validate a murine Pcdh19 null allele in which a β-Geo reporter cassette is expressed under the control of the endogenous promoter. Analysis of β-Geo reporter activity revealed widespread but restricted expression of PCDH19 in embryonic, postnatal and adult brains. No gross morphological defects were identified in Pcdh19+/β-Geo and Pcdh19Y/β-Geo brains and the location of Pcdh19 null cells was normal. However, in vitro migration assays revealed that the motility of Pcdh19 null neurons was significantly elevated, potentially contributing to pathogenesis in patients with PCDH19 mutations. Overall our initial characterization of Pcdh19+/β-Geo, Pcdh19β-Geo/β-Geo and Pcdh19Y/β-Geomice reveals that despite widespread expression of Pcdh19 in the CNS and its role in human epilepsy, its function in mice is not essential for brain development.

Neurodevelopmental disorders such as intellectual disability (ID), epilepsy, autism spectrum disorders (ASDs), schizophrenia (SZ), and bipolar disorder (BD) include cognitive, neurological, and/or psychiatric dysfunction and can be caused by impairment of the brain during development. These disorders are complex and highly heterogeneous and often coexist with other types of co-comorbidities. Mutations in genes encoding for cell adhesion molecules, specifically the delta-2-protocadherin (. 2-PCDH) family have been shown to be associated with a number of these disorders. These genes have crucial roles in early brain development, including neuronal migration, synaptogenesis, and axonal growth. Currently, little is known about how mutations in the protocadherin gene family contribute to the underlying pathogenesis of neurodevelopmental disorders. This review will discusses different neurodevelopmental disorders and the role of their respective associated published ?2-PCDHs mutations, in particular PCDH19, including new insights into novel mutations potentially contributing to these diseases. Refereed/Peer-reviewed

Our previous studies have shown that nutritional zinc restriction exacerbates airway inflammation accompanied by an increase in caspase-3 activation and an accumulation of apoptotic epithelial cells in the bronchioles of the mice. Normally, apoptotic cells are rapidly cleared by macrophage efferocytosis, limiting any secondary necrosis and inflammation. We therefore hypothesized that zinc deficiency is not only pro-apoptotic but also impairs macrophage efferocytosis. Impaired efferocytic clearance of apoptotic epithelial cells by alveolar macrophages occurs in chronic obstructive pulmonary disease (COPD), cigarette-smoking and other lung inflammatory diseases. We now show that zinc is a factor in impaired macrophage efferocytosis in COPD. Concentrations of zinc were significantly reduced in the supernatant of bronchoalveolar lavage fluid of patients with COPD who were current smokers, compared to healthy controls, smokers or COPD patients not actively smoking. Lavage zinc was positively correlated with AM efferocytosis and there was decreased efferocytosis in macrophages depleted of Zn in vitro by treatment with the membrane-permeable zinc chelator TPEN. Organ and cell Zn homeostasis are mediated by two families of membrane ZIP and ZnT proteins. Macrophages of mice null for ZIP1 had significantly lower intracellular zinc and efferocytosis capability, suggesting ZIP1 may play an important role. We investigated further using the human THP-1 derived macrophage cell line, with and without zinc chelation by TPEN to mimic zinc deficiency. There was no change in ZIP1 mRNA levels by TPEN but a significant 3-fold increase in expression of another influx transporter ZIP2, consistent with a role for ZIP2 in maintaining macrophage Zn levels. Both ZIP1 and ZIP2 proteins were localized to the plasma membrane and cytoplasm in normal human lung alveolar macrophages. We propose that zinc homeostasis in macrophages involves the coordinated action of ZIP1 and ZIP2 transporters responding differently to zinc deficiency signals and that these play important roles in macrophage efferocytosis. Refereed/Peer-reviewed

Loss-of-function mutations in UPF3B result in variable clinical presentations including intellectual disability (ID, syndromic and non-syndromic), autism, childhood onset schizophrenia and attention deficit hyperactivity disorder. UPF3B is a core member of the nonsense-mediatedmRNAdecay (NMD) pathway that functions to rapidly degrade transcripts with premature termination codons (PTCs). Traditionally identified in thousands of human diseases, PTCs were recently also found to be part of 'normal' genetic variation in human populations. Furthermore, many human transcripts have naturally occurring regulatory features compatible with 'endogenous'PTCsstrongly suggesting roles ofNMDbeyondPTCmRNAcontrol. In this study,weinvestigated the role of Upf3b andNMD in neural cells.Weprovide evidence that suggests Upf3b-dependentNMD(Upf3b-NMD) is regulated at multiple levels during development including regulation of expression and sub-cellular localization of Upf3b. Furthermore, complementary expression of Upf3b, Upf3a and Stau1 stratify the developing dorsal telencephalon, suggesting that alternativeNMD,andthe related Staufen1-mediatedmRNAdecay (SMD) pathways are differentially employed. A loss of Upf3b-NMD in neural progenitor cells (NPCs) resulted in the expansion of cell numbers at the expense of their differentiation. In primary hippocampal neurons, loss of Upf3b-NMD resulted in subtle neurite growth effects. Our data suggest that the cellular consequences of loss of Upf3b-NMD can be explained in-part by changes in expression of key NMD-feature containing transcripts, which are commonly deregulated also in patients with UPF3B mutations. Our research identifies novel pathological mechanisms of UPF3B mutations and at least partly explains the clinical phenotype of UPF3B patients. Refereed/Peer-reviewed