Genetic and chemotherapeutic influences on germline hypermutation.

Mutations in the germline generates all evolutionary genetic variation and is a cause of genetic disease. Parental age is the primary determinant of the number of new germline mutations in an individual's genome1,2. Here we analysed the genome-wide sequences of 21,879 families with rare genetic diseases and identified 12 individuals with a hypermutated genome with between two and seven times more de novo single-nucleotide variants than expected. In most families (9 out of 12), the excess mutations came from the father. Two families had genetic drivers of germline hypermutation, with fathers carrying damaging genetic variation in DNA-repair genes. For five of the families, paternal exposure to chemotherapeutic agents before conception was probably a key driver of hypermutation. Our results suggest that the germline is well protected from mutagenic effects, hypermutation is rare, the number of excess mutations is relatively modest and most individuals with a hypermutated genome will not have a genetic disease.

Evidence for 28 genetic disorders discovered by combining healthcare and research data.

De novo mutations in protein-coding genes are a well-established cause of developmental disorders1. However, genes known to be associated with developmental disorders account for only a minority of the observed excess of such de novo mutations1,2. Here, to identify previously undescribed genes associated with developmental disorders, we integrate healthcare and research exome-sequence data from 31,058 parent-offspring trios of individuals with developmental disorders, and develop a simulation-based statistical test to identify gene-specific enrichment of de novo mutations. We identified 285 genes that were significantly associated with developmental disorders, including 28 that had not previously been robustly associated with developmental disorders. Although we detected more genes associated with developmental disorders, much of the excess of de novo mutations in protein-coding genes remains unaccounted for. Modelling suggests that more than 1,000 genes associated with developmental disorders have not yet been described, many of which are likely to be less penetrant than the currently known genes. Research access to clinical diagnostic datasets will be critical for completing the map of genes associated with developmental disorders.

Autosomal dominant ApoA4 mutations present as tubulointerstitial kidney disease with medullary amyloidosis.

Sporadic cases of apolipoprotein A-IV medullary amyloidosis have been reported. Here we describe five families found to have autosomal dominant medullary amyloidosis due to two different pathogenic APOA4 variants. A large family with autosomal dominant chronic kidney disease (CKD) and bland urinary sediment underwent whole genome sequencing with identification of a chr11:116692578 G>C (hg19) variant encoding the missense mutation p.L66V of the ApoA4 protein. We identified two other distantly related families from our registry with the same variant and two other distantly related families with a chr11:116693454 C>T (hg19) variant encoding the missense mutation p.D33N. Both mutations are unique to affected families, evolutionarily conserved and predicted to expand the amyloidogenic hotspot in the ApoA4 structure. Clinically affected individuals suffered from CKD with a bland urinary sediment and a mean age for kidney failure of 64.5 years. Genotyping identified 48 genetically affected individuals; 44 individuals had an estimated glomerular filtration rate (eGFR) under 60 ml/min/1.73 m2, including all 25 individuals with kidney failure. Significantly, 11 of 14 genetically unaffected individuals had an eGFR over 60 ml/min/1.73 m2. Fifteen genetically affected individuals presented with higher plasma ApoA4 concentrations. Kidney pathologic specimens from four individuals revealed amyloid deposits limited to the medulla, with the mutated ApoA4 identified by mass-spectrometry as the predominant amyloid constituent in all three available biopsies. Thus, ApoA4 mutations can cause autosomal dominant medullary amyloidosis, with marked amyloid deposition limited to the kidney medulla and presenting with autosomal dominant CKD with a bland urinary sediment. Diagnosis relies on a careful family history, APOA4 sequencing and pathologic studies.

De novo mutations in regulatory elements in neurodevelopmental disorders.

We previously estimated that 42% of patients with severe developmental disorders carry pathogenic de novo mutations in coding sequences. The role of de novo mutations in regulatory elements affecting genes associated with developmental disorders, or other genes, has been essentially unexplored. We identified de novo mutations in three classes of putative regulatory elements in almost 8,000 patients with developmental disorders. Here we show that de novo mutations in highly evolutionarily conserved fetal brain-active elements are significantly and specifically enriched in neurodevelopmental disorders. We identified a significant twofold enrichment of recurrently mutated elements. We estimate that, genome-wide, 1-3% of patients without a diagnostic coding variant carry pathogenic de novo mutations in fetal brain-active regulatory elements and that only 0.15% of all possible mutations within highly conserved fetal brain-active elements cause neurodevelopmental disorders with a dominant mechanism. Our findings represent a robust estimate of the contribution of de novo mutations in regulatory elements to this genetically heterogeneous set of disorders, and emphasize the importance of combining functional and evolutionary evidence to identify regulatory causes of genetic disorders.

EyeG2P: an automated variant filtering approach improves efficiency of diagnostic genomic testing for inherited ophthalmic disorders.

BACKGROUND: Genomic variant prioritisation is one of the most significant bottlenecks to mainstream genomic testing in healthcare. Tools to improve precision while ensuring high recall are critical to successful mainstream clinical genomic testing, in particular for whole genome sequencing where millions of variants must be considered for each patient. METHODS: We developed EyeG2P, a publicly available database and web application using the Ensembl Variant Effect Predictor. EyeG2P is tailored for efficient variant prioritisation for individuals with inherited ophthalmic conditions. We assessed the sensitivity of EyeG2P in 1234 individuals with a broad range of eye conditions who had previously received a confirmed molecular diagnosis through routine genomic diagnostic approaches. For a prospective cohort of 83 individuals, we assessed the precision of EyeG2P in comparison with routine diagnostic approaches. For 10 additional individuals, we assessed the utility of EyeG2P for whole genome analysis. RESULTS: EyeG2P had 99.5% sensitivity for genomic variants previously identified as clinically relevant through routine diagnostic analysis (n=1234 individuals). Prospectively, EyeG2P enabled a significant increase in precision (35% on average) in comparison with routine testing strategies (p<0.001). We demonstrate that incorporation of EyeG2P into whole genome sequencing analysis strategies can reduce the number of variants for analysis to six variants, on average, while maintaining high diagnostic yield. CONCLUSION: Automated filtering of genomic variants through EyeG2P can increase the efficiency of diagnostic testing for individuals with a broad range of inherited ophthalmic disorders.

Pathogenicity and selective constraint on variation near splice sites.

Mutations that perturb normal pre-mRNA splicing are significant contributors to human disease. We used exome sequencing data from 7833 probands with developmental disorders (DDs) and their unaffected parents, as well as more than 60,000 aggregated exomes from the Exome Aggregation Consortium, to investigate selection around the splice sites and quantify the contribution of splicing mutations to DDs. Patterns of purifying selection, a deficit of variants in highly constrained genes in healthy subjects, and excess de novo mutations in patients highlighted particular positions within and around the consensus splice site of greater functional relevance. By using mutational burden analyses in this large cohort of proband-parent trios, we could estimate in an unbiased manner the relative contributions of mutations at canonical dinucleotides (73%) and flanking noncanonical positions (27%), and calculate the positive predictive value of pathogenicity for different classes of mutations. We identified 18 patients with likely diagnostic de novo mutations in dominant DD-associated genes at noncanonical positions in splice sites. We estimate 35%-40% of pathogenic variants in noncanonical splice site positions are missing from public databases.

Impact of Parental Illness and Injury on Pediatric Disorders of Gut-Brain Interaction.

OBJECTIVE: To evaluate the relationship between parental injury and illness and disorders of gut-brain interaction (DGBI) in children. STUDY DESIGN: A self-controlled case series using data from the Military Health System Data Repository compared International Classification of Diseases, Ninth Revision-identified DGBI-related outpatient visits and prescriptions in 442 651 children aged 3-16 years in the 2 years before and the 2 years after the injury and/or illness of their military parent. Negative binomial regression was used to compare visit rates for constipation, fecal incontinence, abdominal pain, irritable bowel syndrome, and a composite of these before and after parental injury and/or illness. Logistic regression, clustered by child, compared the odds of stooling agent and antispasmodic prescription before and after parental injury and/or illness. RESULTS: In the 2 years following parental injury and/or illness, children had increased visits for DGBIs (adjusted incidence rate ratio [aIRR] 1.09; 95% CI 1.07-1.10), constipation (aIRR 1.07; 95% CI 1.04-1.10), abdominal pain (aIRR 1.09; 95% CI 1.07-1.12), and irritable bowel syndrome (aIRR 1.37; 95% CI 1.19-1.58). Following parental injury and/or illness, the odds of stooling agent prescription decreased (aOR 0.95; 95% CI 0.93-0.97) and the odds of antispasmodic prescription increased (aOR 1.26; 95% CI 1.18-1.36). CONCLUSIONS: Parental injury and/or illness is associated with increased healthcare use for DGBIs. Parental health should be considered by clinicians when assessing DGBIs, counseling patients, and formulating treatment plans.

TNFα signals through specialized factories where responsive coding and miRNA genes are transcribed.

Tumour necrosis factor alpha (TNFα) is a potent cytokine that signals through nuclear factor kappa B (NFκB) to activate a subset of human genes. It is usually assumed that this involves RNA polymerases transcribing responsive genes wherever they might be in the nucleus. Using primary human endothelial cells, variants of chromosome conformation capture (including 4C and chromatin interaction analysis with paired-end tag sequencing), and fluorescence in situ hybridization to detect single nascent transcripts, we show that TNFα induces responsive genes to congregate in discrete 'NFκB factories'. Some factories further specialize in transcribing responsive genes encoding micro-RNAs that target downregulated mRNAs. We expect all signalling pathways to contain this extra leg, where responding genes are transcribed in analogous specialized factories.

Dynamic changes in the epigenomic landscape regulate human organogenesis and link to developmental disorders.

How the genome activates or silences transcriptional programmes governs organ formation. Little is known in human embryos undermining our ability to benchmark the fidelity of stem cell differentiation or cell programming, or interpret the pathogenicity of noncoding variation. Here, we study histone modifications across thirteen tissues during human organogenesis. We integrate the data with transcription to build an overview of how the human genome differentially regulates alternative organ fates including by repression. Promoters from nearly 20,000 genes partition into discrete states. Key developmental gene sets are actively repressed outside of the appropriate organ without obvious bivalency. Candidate enhancers, functional in zebrafish, allow imputation of tissue-specific and shared patterns of transcription factor binding. Overlaying more than 700 noncoding mutations from patients with developmental disorders allows correlation to unanticipated target genes. Taken together, the data provide a comprehensive genomic framework for investigating normal and abnormal human development.

Contribution of retrotransposition to developmental disorders.

Mobile genetic Elements (MEs) are segments of DNA which can copy themselves and other transcribed sequences through the process of retrotransposition (RT). In humans several disorders have been attributed to RT, but the role of RT in severe developmental disorders (DD) has not yet been explored. Here we identify RT-derived events in 9738 exome sequenced trios with DD-affected probands. We ascertain 9 de novo MEs, 4 of which are likely causative of the patient's symptoms (0.04%), as well as 2 de novo gene retroduplications. Beyond identifying likely diagnostic RT events, we estimate genome-wide germline ME mutation rate and selective constraint and demonstrate that coding RT events have signatures of purifying selection equivalent to those of truncating mutations. Overall, our analysis represents a comprehensive interrogation of the impact of retrotransposition on protein coding genes and a framework for future evolutionary and disease studies.

Quantifying the contribution of recessive coding variation to developmental disorders.

We estimated the genome-wide contribution of recessive coding variation in 6040 families from the Deciphering Developmental Disorders study. The proportion of cases attributable to recessive coding variants was 3.6% in patients of European ancestry, compared with 50% explained by de novo coding mutations. It was higher (31%) in patients with Pakistani ancestry, owing to elevated autozygosity. Half of this recessive burden is attributable to known genes. We identified two genes not previously associated with recessive developmental disorders, KDM5B and EIF3F, and functionally validated them with mouse and cellular models. Our results suggest that recessive coding variants account for a small fraction of currently undiagnosed nonconsanguineous individuals, and that the role of noncoding variants, incomplete penetrance, and polygenic mechanisms need further exploration.

Towards programming immune tolerance through geometric manipulation of phosphatidylserine.

The possibility of engineering the immune system in a targeted fashion using biomaterials such as nanoparticles has made considerable headway in recent years. However, little is known as to how modulating the spatial presentation of a ligand augments downstream immune responses. In this report we show that geometric manipulation of phosphatidylserine (PS) through fabrication on rod-shaped PLGA nanoparticles robustly dampens inflammatory responses from innate immune cells while promoting T regulatory cell abundance by impeding effector T cell expansion. This response depends on the geometry of PS presentation as both PS liposomes and 1 micron cylindrical PS-PLGA particles are less potent signal inducers than 80 × 320 nm rod-shaped PS-PLGA particles for an equivalent dose of PS. We show that this immune tolerizing effect can be co-opted for therapeutic benefit in a mouse model of multiple sclerosis and an assay of organ rejection using a mixed lymphocyte reaction with primary human immune cells. These data provide evidence that geometric manipulation of a ligand via biomaterials may enable more efficient and tunable programming of cellular signaling networks for therapeutic benefit in a variety of disease states, including autoimmunity and organ rejection, and thus should be an active area of further research.