Application of multiple mosaic callers improves post-zygotic mutation detection from exome sequencing data.

PURPOSE: The gold standard for identification of post-zygotic variants (PZVs) is droplet digital PCR (ddPCR) or high-depth sequencing across multiple tissues types. These approaches are yet to be systematically implemented for monogenic disorders. We developed PZV detection pipelines for correct classification of de novo variants. METHOD: Our pipelines detect PZV in parents (gonosomal mosaicism "pGoM") and children (somatic mosaicism, "M3"). We applied them to research exome sequencing (ES) data from The Australian Cerebral Palsy Biobank (ACPB, n=145 trios) and Simons Simplex Collection (SSC, n=405 families). Candidate mosaic variants were validated using deep amplicon sequencing or ddPCR. RESULTS: 69.2% (M3trio), 63.9% (M3single) and 92.7% (pGoM) of detected variants were validated, with 48.6%, 56.7% and 26.2% of variants respectively meeting strict criteria for mosaicism. In the ACPB, 16.6% of probands and 20.7% of parents had at least one true positive somatic or pGoM variant respectively. A large proportion of PZVs detected in SSC parents (79.8%) and child (94.5%) were not previously reported. We reclassified 3.7-8.0% of germline de novo variants as mosaic. CONCLUSION: Many PZVs were incorrectly classified as germline variants or missed by previous approaches. Systematic application of our pipelines could increase genetic diagnostic rate, improve estimates of recurrence risk in families, and benefit novel disease gene identification.

Compromised transcription-mRNA export factor THOC2 causes R-loop accumulation, DNA damage and adverse neurodevelopment.

We implicated the X-chromosome THOC2 gene, which encodes the largest subunit of the highly-conserved TREX (Transcription-Export) complex, in a clinically complex neurodevelopmental disorder with intellectual disability as the core phenotype. To study the molecular pathology of this essential eukaryotic gene, we generated a mouse model based on a hypomorphic Thoc2 exon 37-38 deletion variant of a patient with ID, speech delay, hypotonia, and microcephaly. The Thoc2 exon 37-38 deletion male (Thoc2Δ/Y) mice recapitulate the core phenotypes of THOC2 syndrome including smaller size and weight, and significant deficits in spatial learning, working memory and sensorimotor functions. The Thoc2Δ/Y mouse brain development is significantly impacted by compromised THOC2/TREX function resulting in R-loop accumulation, DNA damage and consequent cell death. Overall, we suggest that perturbed R-loop homeostasis, in stem cells and/or differentiated cells in mice and the patient, and DNA damage-associated functional alterations are at the root of THOC2 syndrome.

The Enemy within: Innate Surveillance-Mediated Cell Death, the Common Mechanism of Neurodegenerative Disease.

Neurodegenerative diseases comprise an array of progressive neurological disorders all characterized by the selective death of neurons in the central nervous system. Although, rare (familial) and common (sporadic) forms can occur for the same disease, it is unclear whether this reflects several distinct pathogenic pathways or the convergence of different causes into a common form of nerve cell death. Remarkably, neurodegenerative diseases are increasingly found to be accompanied by activation of the innate immune surveillance system normally associated with pathogen recognition and response. Innate surveillance is the cell's quality control system for the purpose of detecting such danger signals and responding in an appropriate manner. Innate surveillance is an "intelligent system," in that the manner of response is relevant to the magnitude and duration of the threat. If possible, the threat is dealt with within the cell in which it is detected, by degrading the danger signal(s) and restoring homeostasis. If this is not successful then an inflammatory response is instigated that is aimed at restricting the spread of the threat by elevating degradative pathways, sensitizing neighboring cells, and recruiting specialized cell types to the site. If the danger signal persists, then the ultimate response can include not only the programmed cell death of the original cell, but the contents of this dead cell can also bring about the death of adjacent sensitized cells. These responses are clearly aimed at destroying the ability of the detected pathogen to propagate and spread. Innate surveillance comprises intracellular, extracellular, non-cell autonomous and systemic processes. Recent studies have revealed how multiple steps in these processes involve proteins that, through their mutation, have been linked to many familial forms of neurodegenerative disease. This suggests that individuals harboring these mutations may have an amplified response to innate-mediated damage in neural tissues, and renders innate surveillance mediated cell death a plausible common pathogenic pathway responsible for neurodegenerative diseases, in both familial and sporadic forms. Here we have assembled evidence in favor of the hypothesis that neurodegenerative disease is the cumulative result of chronic activation of the innate surveillance pathway, triggered by endogenous or environmental danger or damage associated molecular patterns in a progressively expanding cascade of inflammation, tissue damage and cell death.