Epigenetic Drifts during Long-Term Intestinal Organoid Culture

Organoids retain the morphological and molecular patterns of their tissue of origin, are self-organizing, relatively simple to handle and accessible to genetic engineering. Thus, they represent an optimal tool for studying the mechanisms of tissue maintenance and aging. Long-term expansion under standard growth conditions, however, is accompanied by changes in the growth pattern and kinetics. As a potential explanation of these alterations, epigenetic drifts in organoid culture have been suggested. Here, we studied histone tri-methylation at lysine 4 (H3K4me3) and 27 (H3K27me3) and transcriptome profiles of intestinal organoids derived from mismatch repair (MMR)-deficient and control mice and cultured for 3 and 20 weeks and compared them with data on their tissue of origin. We found that, besides the expected changes in short-term culture, the organoids showed profound changes in their epigenomes also during the long-term culture. The most prominent were epigenetic gene activation by H3K4me3 recruitment to previously unmodified genes and by H3K27me3 loss from originally bivalent genes. We showed that a long-term culture is linked to broad transcriptional changes that indicate an ongoing maturation and metabolic adaptation process. This process was disturbed in MMR-deficient mice, resulting in endoplasmic reticulum (ER) stress and Wnt activation. Our results can be explained in terms of a mathematical model assuming that epigenetic changes during a long-term culture involve DNA demethylation that ceases if the metabolic adaptation is disturbed

Point Mutations in GLI3 Lead to Misregulation of its Subcellular Localization

Background Mutations in the transcription factor GLI3, a downstream target of Sonic Hedgehog (SHH) signaling, are responsible for the development of malformation syndromes such as Greig-cephalopolysyndactyly-syndrome (GCPS), or Pallister-Hall-syndrome (PHS). Mutations that lead to loss of function of the protein and to haploinsufficiency cause GCPS, while truncating mutations that result in constitutive repressor function of GLI3 lead to PHS. As an exception, some point mutations in the C-terminal part of GLI3 observed in GCPS patients have so far not been linked to loss of function. We have shown recently that protein phosphatase 2A (PP2A) regulates the nuclear localization and transcriptional activity a of GLI3 function. Principal Findings We have shown recently that protein phosphatase 2A (PP2A) and the ubiquitin ligase MID1 regulate the nuclear localization and transcriptional activity of GLI3. Here we show mapping of the functional interaction between the MID1-α4-PP2A complex and GLI3 to a region between amino acid 568-1100 of GLI3. Furthermore we demonstrate that GCPS-associated point mutations, that are located in that region, lead to misregulation of the nuclear GLI3-localization and transcriptional activity. GLI3 phosphorylation itself however appears independent of its localization and remains untouched by either of the point mutations and by PP2A-activity, which suggests involvement of an as yet unknown GLI3 interaction partner, the phosphorylation status of which is regulated by PP2A activity, in the control of GLI3 subcellular localization and activity. Conclusions The present findings provide an explanation for the pathogenesis of GCPS in patients carrying C-terminal point mutations, and close the gap in our understanding of how GLI3-genotypes give rise to particular phenotypes. Furthermore, they provide a molecular explanation for the phenotypic overlap between Opitz syndrome patients with dysregulated PP2A-activity and syndromes caused by GLI3-mutations

Behavioural and functional evidence revealing the role of RBFOX1 variation in multiple psychiatric disorders and traits

Common variation in the gene encoding the neuron-specific RNA splicing factor RNA Binding Fox-1 Homolog 1 (RBFOX1) has been identified as a risk factor for several psychiatric conditions, and rare genetic variants have been found causal for autism spectrum disorder (ASD). Here, we explored the genetic landscape of RBFOX1 more deeply, integrating evidence from existing and new human studies as well as studies in Rbfox1 knockout mice. Mining existing data from large-scale studies of human common genetic variants, we confirmed gene-based and genome-wide association of RBFOX1 with risk tolerance, major depressive disorder and schizophrenia. Data on six mental disorders revealed copy number losses and gains to be more frequent in ASD cases than in controls. Consistently, RBFOX1 expression appeared decreased in post-mortem frontal and temporal cortices of individuals with ASD and prefrontal cortex of individuals with schizophrenia. Brain-functional MRI studies demonstrated that carriers of a common RBFOX1 variant, rs6500744, displayed increased neural reactivity to emotional stimuli, reduced prefrontal processing during cognitive control, and enhanced fear expression after fear conditioning, going along with increased avoidance behaviour. Investigating Rbfox1 neuron-specific knockout mice allowed us to further specify the role of this gene in behaviour. The model was characterised by pronounced hyperactivity, stereotyped behaviour, impairments in fear acquisition and extinction, reduced social interest, and lack of aggression; it provides excellent construct and face validity as an animal model of ASD. In conclusion, convergent translational evidence shows that common variants in RBFOX1 are associated with a broad spectrum of psychiatric traits and disorders, while rare genetic variation seems to expose to early-onset neurodevelopmental psychiatric disorders with and without developmental delay like ASD, in particular. Studying the pleiotropic nature of RBFOX1 can profoundly enhance our understanding of mental disorder vulnerability

Prevalence and architecture of de novo mutations in developmental disorders.

The genomes of individuals with severe, undiagnosed developmental disorders are enriched in damaging de novo mutations (DNMs) in developmentally important genes. Here we have sequenced the exomes of 4,293 families containing individuals with developmental disorders, and meta-analysed these data with data from another 3,287 individuals with similar disorders. We show that the most important factors influencing the diagnostic yield of DNMs are the sex of the affected individual, the relatedness of their parents, whether close relatives are affected and the parental ages. We identified 94 genes enriched in damaging DNMs, including 14 that previously lacked compelling evidence of involvement in developmental disorders. We have also characterized the phenotypic diversity among these disorders. We estimate that 42% of our cohort carry pathogenic DNMs in coding sequences; approximately half of these DNMs disrupt gene function and the remainder result in altered protein function. We estimate that developmental disorders caused by DNMs have an average prevalence of 1 in 213 to 1 in 448 births, depending on parental age. Given current global demographics, this equates to almost 400,000 children born per year

Heterozygous Variants in KMT2E Cause a Spectrum of Neurodevelopmental Disorders and Epilepsy.

We delineate a KMT2E-related neurodevelopmental disorder on the basis of 38 individuals in 36 families. This study includes 31 distinct heterozygous variants in KMT2E (28 ascertained from Matchmaker Exchange and three previously reported), and four individuals with chromosome 7q22.2-22.23 microdeletions encompassing KMT2E (one previously reported). Almost all variants occurred de novo, and most were truncating. Most affected individuals with protein-truncating variants presented with mild intellectual disability. One-quarter of individuals met criteria for autism. Additional common features include macrocephaly, hypotonia, functional gastrointestinal abnormalities, and a subtle facial gestalt. Epilepsy was present in about one-fifth of individuals with truncating variants and was responsive to treatment with anti-epileptic medications in almost all. More than 70% of the individuals were male, and expressivity was variable by sex; epilepsy was more common in females and autism more common in males. The four individuals with microdeletions encompassing KMT2E generally presented similarly to those with truncating variants, but the degree of developmental delay was greater. The group of four individuals with missense variants in KMT2E presented with the most severe developmental delays. Epilepsy was present in all individuals with missense variants, often manifesting as treatment-resistant infantile epileptic encephalopathy. Microcephaly was also common in this group. Haploinsufficiency versus gain-of-function or dominant-negative effects specific to these missense variants in KMT2E might explain this divergence in phenotype, but requires independent validation. Disruptive variants in KMT2E are an under-recognized cause of neurodevelopmental abnormalities

Bi-allelic Loss-of-Function CACNA1B Mutations in Progressive Epilepsy-Dyskinesia.

The occurrence of non-epileptic hyperkinetic movements in the context of developmental epileptic encephalopathies is an increasingly recognized phenomenon. Identification of causative mutations provides an important insight into common pathogenic mechanisms that cause both seizures and abnormal motor control. We report bi-allelic loss-of-function CACNA1B variants in six children from three unrelated families whose affected members present with a complex and progressive neurological syndrome. All affected individuals presented with epileptic encephalopathy, severe neurodevelopmental delay (often with regression), and a hyperkinetic movement disorder. Additional neurological features included postnatal microcephaly and hypotonia. Five children died in childhood or adolescence (mean age of death: 9 years), mainly as a result of secondary respiratory complications. CACNA1B encodes the pore-forming subunit of the pre-synaptic neuronal voltage-gated calcium channel Cav2.2/N-type, crucial for SNARE-mediated neurotransmission, particularly in the early postnatal period. Bi-allelic loss-of-function variants in CACNA1B are predicted to cause disruption of Ca2+ influx, leading to impaired synaptic neurotransmission. The resultant effect on neuronal function is likely to be important in the development of involuntary movements and epilepsy. Overall, our findings provide further evidence for the key role of Cav2.2 in normal human neurodevelopment.MAK is funded by an NIHR Research Professorship and receives funding from the Wellcome Trust, Great Ormond Street Children's Hospital Charity, and Rosetrees Trust. E.M. received funding from the Rosetrees Trust (CD-A53) and Great Ormond Street Hospital Children's Charity. K.G. received funding from Temple Street Foundation. A.M. is funded by Great Ormond Street Hospital, the National Institute for Health Research (NIHR), and Biomedical Research Centre. F.L.R. and D.G. are funded by Cambridge Biomedical Research Centre. K.C. and A.S.J. are funded by NIHR Bioresource for Rare Diseases. The DDD Study presents independent research commissioned by the Health Innovation Challenge Fund (grant number HICF-1009-003), a parallel funding partnership between the Wellcome Trust and the Department of Health, and the Wellcome Trust Sanger Institute (grant number WT098051). We acknowledge support from the UK Department of Health via the NIHR comprehensive Biomedical Research Centre award to Guy's and St. Thomas' National Health Service (NHS) Foundation Trust in partnership with King's College London. This research was also supported by the NIHR Great Ormond Street Hospital Biomedical Research Centre. J.H.C. is in receipt of an NIHR Senior Investigator Award. The research team acknowledges the support of the NIHR through the Comprehensive Clinical Research Network. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, Department of Health, or Wellcome Trust. E.R.M. acknowledges support from NIHR Cambridge Biomedical Research Centre, an NIHR Senior Investigator Award, and the University of Cambridge has received salary support in respect of E.R.M. from the NHS in the East of England through the Clinical Academic Reserve. I.E.S. is supported by the National Health and Medical Research Council of Australia (Program Grant and Practitioner Fellowship)