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)

Functional annotation of variants of the BRCA2 gene via locally haploid human pluripotent stem cells

Mutations in the BRCA2 gene are associated with sporadic and familial cancer, cause genomic instability and sensitize cancer cells to inhibition by the poly(ADP-ribose) polymerase (PARP). Here we show that human pluripotent stem cells (hPSCs) with one copy of BRCA2 deleted can be used to annotate variants of this gene and to test their sensitivities to PARP inhibition. By using Cas9 to edit the functional BRCA2 allele in the locally haploid hPSCs and in fibroblasts differentiated from them, we characterized essential regions in the gene to identify permissive and loss-of-function mutations. We also used Cas9 to directly test the function of individual amino acids, including amino acids encoded by clinical BRCA2 variants of uncertain significance, and identified alleles that are sensitive to PARP inhibitors used as a standard of care in BRCA2-deficient cancers. Locally haploid human pluripotent stem cells can facilitate detailed structure-function analyses of genes and the rapid functional evaluation of clinically observed mutations

Peptide-mediated delivery of CRISPR enzymes for the efficient editing of primary human lymphocytes

CRISPR-mediated genome editing of primary human lymphocytes is typically carried out via electroporation, which can be cytotoxic, cumbersome and costly. Here we show that the yields of edited primary human lymphocytes can be increased substantially by delivering a CRISPR ribonucleoprotein mixed with an amphiphilic peptide identified through screening. We evaluated the performance of this simple delivery method by knocking out genes in T cells, B cells and natural killer cells via the delivery of Cas9 or Cas12a ribonucleoproteins or an adenine base editor. We also show that peptide-mediated ribonucleoprotein delivery paired with an adeno-associated-virus-mediated homology-directed repair template can introduce a chimaeric antigen receptor gene at the T-cell receptor α constant locus, and that the engineered cells display antitumour potency in mice. The method is minimally perturbative, does not require dedicated hardware, and is compatible with multiplexed editing via sequential delivery, which minimizes the risk of genotoxicity. The peptide-mediated intracellular delivery of ribonucleoproteins may facilitate the manufacturing of engineered T cells

Genetic diagnosis of developmental disorders in the DDD study:a scalable analysis of genome-wide research data

SummaryBackgroundHuman genome sequencing has transformed our understanding of genomic variation and its relevance to health and disease, and is now starting to enter clinical practice for the diagnosis of rare diseases. The question of whether and how some categories of genomic findings should be shared with individual research participants is currently a topic of international debate, and development of robust analytical workflows to identify and communicate clinically relevant variants is paramount.MethodsThe Deciphering Developmental Disorders (DDD) study has developed a UK-wide patient recruitment network involving over 180 clinicians across all 24 regional genetics services, and has performed genome-wide microarray and whole exome sequencing on children with undiagnosed developmental disorders and their parents. After data analysis, pertinent genomic variants were returned to individual research participants via their local clinical genetics team.FindingsAround 80 000 genomic variants were identified from exome sequencing and microarray analysis in each individual, of which on average 400 were rare and predicted to be protein altering. By focusing only on de novo and segregating variants in known developmental disorder genes, we achieved a diagnostic yield of 27% among 1133 previously investigated yet undiagnosed children with developmental disorders, whilst minimising incidental findings. In families with developmentally normal parents, whole exome sequencing of the child and both parents resulted in a 10-fold reduction in the number of potential causal variants that needed clinical evaluation compared to sequencing only the child. Most diagnostic variants identified in known genes were novel and not present in current databases of known disease variation.InterpretationImplementation of a robust translational genomics workflow is achievable within a large-scale rare disease research study to allow feedback of potentially diagnostic findings to clinicians and research participants. Systematic recording of relevant clinical data, curation of a gene–phenotype knowledge base, and development of clinical decision support software are needed in addition to automated exclusion of almost all variants, which is crucial for scalable prioritisation and review of possible diagnostic variants. However, the resource requirements of development and maintenance of a clinical reporting system within a research setting are substantial.FundingHealth Innovation Challenge Fund, a parallel funding partnership between the Wellcome Trust and the UK Department of Health

Species- and site-specific genome editing in complex bacterial communities.

Understanding microbial gene functions relies on the application of experimental genetics in cultured microorganisms. However, the vast majority of bacteria and archaea remain uncultured, precluding the application of traditional genetic methods to these organisms and their interactions. Here, we characterize and validate a generalizable strategy for editing the genomes of specific organisms in microbial communities. We apply environmental transformation sequencing (ET-seq), in which nontargeted transposon insertions are mapped and quantified following delivery to a microbial community, to identify genetically tractable constituents. Next, DNA-editing all-in-one RNA-guided CRISPR-Cas transposase (DART) systems for targeted DNA insertion into organisms identified as tractable by ET-seq are used to enable organism- and locus-specific genetic manipulation in a community context. Using a combination of ET-seq and DART in soil and infant gut microbiota, we conduct species- and site-specific edits in several bacteria, measure gene fitness in a nonmodel bacterium and enrich targeted species. These tools enable editing of microbial communities for understanding and control

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

Monitoring SARS-CoV-2 incidence and seroconversion among university students and employees: a longitudinal cohort study in California, June–August 2020

ObjectivesTo identify incident SARS-CoV-2 infections and inform effective mitigation strategies in university settings, we piloted an integrated symptom and exposure monitoring and testing system among a cohort of university students and employees.DesignProspective cohort study.SettingA public university in California from June to August 2020.Participants2180 university students and 738 university employees.Primary outcome measuresAt baseline and endline, we tested participants for active SARS-CoV-2 infection via quantitative PCR (qPCR) test and collected blood samples for antibody testing. Participants received notifications to complete additional qPCR tests throughout the study if they reported symptoms or exposures in daily surveys or were selected for surveillance testing. Viral whole genome sequencing was performed on positive qPCR samples, and phylogenetic trees were constructed with these genomes and external genomes.ResultsOver the study period, 57 students (2.6%) and 3 employees (0.4%) were diagnosed with SARS-CoV-2 infection via qPCR test. Phylogenetic analyses revealed that a super-spreader event among undergraduates in congregate housing accounted for at least 48% of cases among study participants but did not spread beyond campus. Test positivity was higher among participants who self-reported symptoms (incidence rate ratio (IRR) 12.7; 95% CI 7.4 to 21.8) or had household exposures (IRR 10.3; 95% CI 4.8 to 22.0) that triggered notifications to test. Most (91%) participants with newly identified antibodies at endline had been diagnosed with incident infection via qPCR test during the study.ConclusionsOur findings suggest that integrated monitoring systems can successfully identify and link at-risk students to SARS-CoV-2 testing. As the study took place before the evolution of highly transmissible variants and widespread availability of vaccines and rapid antigen tests, further research is necessary to adapt and evaluate similar systems in the present context