A point mutation in the ion conduction pore of AMPA receptor GRIA3 causes dramatically perturbed sleep patterns as well as intellectual disability.

The discovery of genetic variants influencing sleep patterns can shed light on the physiological processes underlying sleep. As part of a large clinical sequencing project, WGS500, we sequenced a family in which the two male children had severe developmental delay and a dramatically disturbed sleep-wake cycle, with very long wake and sleep durations, reaching up to 106-h awake and 48-h asleep. The most likely causal variant identified was a novel missense variant in the X-linked GRIA3 gene, which has been implicated in intellectual disability. GRIA3 encodes GluA3, a subunit of AMPA-type ionotropic glutamate receptors (AMPARs). The mutation (A653T) falls within the highly conserved transmembrane domain of the ion channel gate, immediately adjacent to the analogous residue in the Grid2 (glutamate receptor) gene, which is mutated in the mouse neurobehavioral mutant, Lurcher. In vitro, the GRIA3(A653T) mutation stabilizes the channel in a closed conformation, in contrast to Lurcher. We introduced the orthologous mutation into a mouse strain by CRISPR-Cas9 mutagenesis and found that hemizygous mutants displayed significant differences in the structure of their activity and sleep compared to wild-type littermates. Typically, mice are polyphasic, exhibiting multiple sleep bouts of sleep several minutes long within a 24-h period. The Gria3A653T mouse showed significantly fewer brief bouts of activity and sleep than the wild-types. Furthermore, Gria3A653T mice showed enhanced period lengthening under constant light compared to wild-type mice, suggesting an increased sensitivity to light. Our results suggest a role for GluA3 channel activity in the regulation of sleep behavior in both mice and humans.

Mosaic PPM1D mutations are associated with predisposition to breast and ovarian cancer.

Improved sequencing technologies offer unprecedented opportunities for investigating the role of rare genetic variation in common disease. However, there are considerable challenges with respect to study design, data analysis and replication. Using pooled next-generation sequencing of 507 genes implicated in the repair of DNA in 1,150 samples, an analytical strategy focused on protein-truncating variants (PTVs) and a large-scale sequencing case-control replication experiment in 13,642 individuals, here we show that rare PTVs in the p53-inducible protein phosphatase PPM1D are associated with predisposition to breast cancer and ovarian cancer. PPM1D PTV mutations were present in 25 out of 7,781 cases versus 1 out of 5,861 controls (P = 1.12 × 10(-5)), including 18 mutations in 6,912 individuals with breast cancer (P = 2.42 × 10(-4)) and 12 mutations in 1,121 individuals with ovarian cancer (P = 3.10 × 10(-9)). Notably, all of the identified PPM1D PTVs were mosaic in lymphocyte DNA and clustered within a 370-base-pair region in the final exon of the gene, carboxy-terminal to the phosphatase catalytic domain. Functional studies demonstrate that the mutations result in enhanced suppression of p53 in response to ionizing radiation exposure, suggesting that the mutant alleles encode hyperactive PPM1D isoforms. Thus, although the mutations cause premature protein truncation, they do not result in the simple loss-of-function effect typically associated with this class of variant, but instead probably have a gain-of-function effect. Our results have implications for the detection and management of breast and ovarian cancer risk. More generally, these data provide new insights into the role of rare and of mosaic genetic variants in common conditions, and the use of sequencing in their identification.

Clinical whole-genome sequencing in severe early-onset epilepsy reveals new genes and improves molecular diagnosis.

In severe early-onset epilepsy, precise clinical and molecular genetic diagnosis is complex, as many metabolic and electro-physiological processes have been implicated in disease causation. The clinical phenotypes share many features such as complex seizure types and developmental delay. Molecular diagnosis has historically been confined to sequential testing of candidate genes known to be associated with specific sub-phenotypes, but the diagnostic yield of this approach can be low. We conducted whole-genome sequencing (WGS) on six patients with severe early-onset epilepsy who had previously been refractory to molecular diagnosis, and their parents. Four of these patients had a clinical diagnosis of Ohtahara Syndrome (OS) and two patients had severe non-syndromic early-onset epilepsy (NSEOE). In two OS cases, we found de novo non-synonymous mutations in the genes KCNQ2 and SCN2A. In a third OS case, WGS revealed paternal isodisomy for chromosome 9, leading to identification of the causal homozygous missense variant in KCNT1, which produced a substantial increase in potassium channel current. The fourth OS patient had a recessive mutation in PIGQ that led to exon skipping and defective glycophosphatidyl inositol biosynthesis. The two patients with NSEOE had likely pathogenic de novo mutations in CBL and CSNK1G1, respectively. Mutations in these genes were not found among 500 additional individuals with epilepsy. This work reveals two novel genes for OS, KCNT1 and PIGQ. It also uncovers unexpected genetic mechanisms and emphasizes the power of WGS as a clinical tool for making molecular diagnoses, particularly for highly heterogeneous disorders.

Unlocking the bottleneck in forward genetics using whole-genome sequencing and identity by descent to isolate causative mutations.

Forward genetics screens with N-ethyl-N-nitrosourea (ENU) provide a powerful way to illuminate gene function and generate mouse models of human disease; however, the identification of causative mutations remains a limiting step. Current strategies depend on conventional mapping, so the propagation of affected mice requires non-lethal screens; accurate tracking of phenotypes through pedigrees is complex and uncertain; out-crossing can introduce unexpected modifiers; and Sanger sequencing of candidate genes is inefficient. Here we show how these problems can be efficiently overcome using whole-genome sequencing (WGS) to detect the ENU mutations and then identify regions that are identical by descent (IBD) in multiple affected mice. In this strategy, we use a modification of the Lander-Green algorithm to isolate causative recessive and dominant mutations, even at low coverage, on a pure strain background. Analysis of the IBD regions also allows us to calculate the ENU mutation rate (1.54 mutations per Mb) and to model future strategies for genetic screens in mice. The introduction of this approach will accelerate the discovery of causal variants, permit broader and more informative lethal screens to be used, reduce animal costs, and herald a new era for ENU mutagenesis.

Next-generation sequencing to dissect hereditary nephrotic syndrome in mice identifies a hypomorphic mutation in Lamb2 and models Pierson's syndrome.

The study of mutations causing the steroid-resistant nephrotic syndrome in children has greatly advanced our understanding of the kidney filtration barrier. In particular, these genetic variants have illuminated the roles of the podocyte, glomerular basement membrane and endothelial cell in glomerular filtration. However, in a significant number of familial and early onset cases, an underlying mutation cannot be identified, indicating that there are likely to be multiple unknown genes with roles in glomerular permeability. We now show how the combination of N-ethyl-N-nitrosourea mutagenesis and next-generation sequencing could be used to identify the range of mutations affecting these pathways. Using this approach, we isolated a novel mouse strain with a viable nephrotic phenotype and used whole-genome sequencing to isolate a causative hypomorphic mutation in Lamb2. This discovery generated a model for one part of the spectrum of human Pierson's syndrome and provides a powerful proof of principle for accelerating gene discovery and improving our understanding of inherited forms of renal disease.

Evolutionary dynamics of Staphylococcus aureus during progression from carriage to disease.

Whole-genome sequencing offers new insights into the evolution of bacterial pathogens and the etiology of bacterial disease. Staphylococcus aureus is a major cause of bacteria-associated mortality and invasive disease and is carried asymptomatically by 27% of adults. Eighty percent of bacteremias match the carried strain. However, the role of evolutionary change in the pathogen during the progression from carriage to disease is incompletely understood. Here we use high-throughput genome sequencing to discover the genetic changes that accompany the transition from nasal carriage to fatal bloodstream infection in an individual colonized with methicillin-sensitive S. aureus. We found a single, cohesive population exhibiting a repertoire of 30 single-nucleotide polymorphisms and four insertion/deletion variants. Mutations accumulated at a steady rate over a 13-mo period, except for a cluster of mutations preceding the transition to disease. Although bloodstream bacteria differed by just eight mutations from the original nasally carried bacteria, half of those mutations caused truncation of proteins, including a premature stop codon in an AraC-family transcriptional regulator that has been implicated in pathogenicity. Comparison with evolution in two asymptomatic carriers supported the conclusion that clusters of protein-truncating mutations are highly unusual. Our results demonstrate that bacterial diversity in vivo is limited but nonetheless detectable by whole-genome sequencing, enabling the study of evolutionary dynamics within the host. Regulatory or structural changes that occur during carriage may be functionally important for pathogenesis; therefore identifying those changes is a crucial step in understanding the biological causes of invasive bacterial disease.

Recessive mutations in SPTBN2 implicate ß-III spectrin in both cognitive and motor development.

ß-III spectrin is present in the brain and is known to be important in the function of the cerebellum. Heterozygous mutations in SPTBN2, the gene encoding ß-III spectrin, cause Spinocerebellar Ataxia Type 5 (SCA5), an adult-onset, slowly progressive, autosomal-dominant pure cerebellar ataxia. SCA5 is sometimes known as "Lincoln ataxia," because the largest known family is descended from relatives of the United States President Abraham Lincoln. Using targeted capture and next-generation sequencing, we identified a homozygous stop codon in SPTBN2 in a consanguineous family in which childhood developmental ataxia co-segregates with cognitive impairment. The cognitive impairment could result from mutations in a second gene, but further analysis using whole-genome sequencing combined with SNP array analysis did not reveal any evidence of other mutations. We also examined a mouse knockout of ß-III spectrin in which ataxia and progressive degeneration of cerebellar Purkinje cells has been previously reported and found morphological abnormalities in neurons from prefrontal cortex and deficits in object recognition tasks, consistent with the human cognitive phenotype. These data provide the first evidence that ß-III spectrin plays an important role in cortical brain development and cognition, in addition to its function in the cerebellum; and we conclude that cognitive impairment is an integral part of this novel recessive ataxic syndrome, Spectrin-associated Autosomal Recessive Cerebellar Ataxia type 1 (SPARCA1). In addition, the identification of SPARCA1 and normal heterozygous carriers of the stop codon in SPTBN2 provides insights into the mechanism of molecular dominance in SCA5 and demonstrates that the cell-specific repertoire of spectrin subunits underlies a novel group of disorders, the neuronal spectrinopathies, which includes SCA5, SPARCA1, and a form of West syndrome.

CoverView: a sequence quality evaluation tool for next generation sequencing data.

Quality assurance and quality control are essential for robust next generation sequencing (NGS). Here we present CoverView, a fast, flexible, user-friendly quality evaluation tool for NGS data. CoverView processes mapped sequencing reads and user-specified regions to report depth of coverage, base and mapping quality metrics with increasing levels of detail from a chromosome-level summary to per-base profiles. CoverView can flag regions that do not fulfil user-specified quality requirements, allowing suboptimal data to be systematically and automatically presented for review. It also provides an interactive graphical user interface (GUI) that can be opened in a web browser and allows intuitive exploration of results. We have integrated CoverView into our accredited clinical cancer predisposition gene testing laboratory that uses the TruSight Cancer Panel (TSCP). CoverView has been invaluable for optimisation and quality control of our testing pipeline, providing transparent, consistent quality metric information and automatic flagging of regions that fall below quality thresholds. We demonstrate this utility with TSCP data from the Genome in a Bottle reference sample, which CoverView analysed in 13 seconds. CoverView uses data routinely generated by NGS pipelines, reads standard input formats, and rapidly creates easy-to-parse output text (.txt) files that are customised by a simple configuration file. CoverView can therefore be easily integrated into any NGS pipeline. CoverView and detailed documentation for its use are freely available at github.com/RahmanTeamDevelopment/CoverView/releases and www.icr.ac.uk/CoverView.

PRO_SELECT: combining structure-based drug design and array-based chemistry for rapid lead discovery. 2. The development of a series of highly potent and selective factor Xa inhibitors.

In silico screening of combinatorial libraries prior to synthesis promises to be a valuable aid to lead discovery. PRO_SELECT, a tool for the virtual screening of libraries for fit to a protein active site, has been used to find novel leads against the serine protease factor Xa. A small seed template was built upon using three iterations of library design, virtual screening, synthesis, and biological testing. Highly potent molecules with selectivity for factor Xa over other serine proteases were rapidly obtained.

Whole-exome sequencing studies of nonfunctioning pituitary adenomas.

CONTEXT: The tumorigenic role of genetic abnormalities in sporadic pituitary nonfunctioning adenomas (NFAs), which usually originate from gonadotroph cells, is unknown. OBJECTIVE: The objective of the study was to identify somatic genetic abnormalities in sporadic pituitary NFAs. DESIGN: Whole-exome sequencing was performed using DNA from 7 pituitary NFAs and leukocyte samples obtained from the same patients. Somatic variants were confirmed by dideoxynucleotide sequencing, and candidate driver genes were assessed in an additional 24 pituitary NFAs. RESULTS: Whole-exome sequencing achieved a high degree of coverage such that approximately 97% of targeted bases were represented by more than 10 base reads; 24 somatic variants were identified and confirmed in the discovery set of 7 pituitary NFAs (mean 3.5 variants/tumor; range 1-7). Approximately 80% of variants occurred as missense single nucleotide variants and the remainder were synonymous changes or small frameshift deletions. Each of the 24 mutations occurred in independent genes with no recurrent mutations. Mutations were not observed in genes previously associated with pituitary tumorigenesis, although somatic variants in putative driver genes including platelet-derived growth factor D (PDGFD), N-myc down-regulated gene family member 4 (NDRG4), and Zipper sterile-α-motif kinase (ZAK) were identified; however, DNA sequence analysis of these in the validation set of 24 pituitary NFAs did not reveal any mutations indicating that these genes are unlikely to contribute significantly in the etiology of sporadic pituitary NFAs. CONCLUSIONS: Pituitary NFAs harbor few somatic mutations consistent with their low proliferation rates and benign nature, but mechanisms other than somatic mutation are likely involved in the etiology of sporadic pituitary NFAs.

B cell survival, surface BCR and BAFFR expression, CD74 metabolism, and CD8- dendritic cells require the intramembrane endopeptidase SPPL2A.

Druggable proteins required for B lymphocyte survival and immune responses are an emerging source of new treatments for autoimmunity and lymphoid malignancy. In this study, we show that mice with an inactivating mutation in the intramembrane protease signal peptide peptidase-like 2A (SPPL2A) unexpectedly exhibit profound humoral immunodeficiency and lack mature B cell subsets, mirroring deficiency of the cytokine B cell-activating factor (BAFF). Accumulation of Sppl2a-deficient B cells was rescued by overexpression of the BAFF-induced survival protein B cell lymphoma 2 (BCL2) but not BAFF and was distinguished by low surface BAFF receptor and IgM and IgD B cell receptors. CD8-negative dendritic cells were also greatly decreased. SPPL2A deficiency blocked the proteolytic processing of CD74 MHC II invariant chain in both cell types, causing dramatic build-up of the p8 product of Cathepsin S and interfering with earlier steps in CD74 endosomal retention and processing. The findings illuminate an important role for the final step in the CD74-MHC II pathway and a new target for protease inhibitor treatment of B cell diseases.

Mutations in AP2S1 cause familial hypocalciuric hypercalcemia type 3.

Adaptor protein-2 (AP2), a central component of clathrin-coated vesicles (CCVs), is pivotal in clathrin-mediated endocytosis, which internalizes plasma membrane constituents such as G protein-coupled receptors (GPCRs). AP2, a heterotetramer of α, ß, µ and σ subunits, links clathrin to vesicle membranes and binds to tyrosine- and dileucine-based motifs of membrane-associated cargo proteins. Here we show that missense mutations of AP2 σ subunit (AP2S1) affecting Arg15, which forms key contacts with dileucine-based motifs of CCV cargo proteins, result in familial hypocalciuric hypercalcemia type 3 (FHH3), an extracellular calcium homeostasis disorder affecting the parathyroids, kidneys and bone. We found AP2S1 mutations in >20% of cases of FHH without mutations in calcium-sensing GPCR (CASR), which cause FHH1. AP2S1 mutations decreased the sensitivity of CaSR-expressing cells to extracellular calcium and reduced CaSR endocytosis, probably through loss of interaction with a C-terminal CaSR dileucine-based motif, whose disruption also decreased intracellular signaling. Thus, our results identify a new role for AP2 in extracellular calcium homeostasis.

Whole-exome sequencing studies of nonhereditary (sporadic) parathyroid adenomas.

CONTEXT: Genetic abnormalities, such as those of multiple endocrine neoplasia type 1 (MEN1) and Cyclin D1 (CCND1) genes, occur in <50% of nonhereditary (sporadic) parathyroid adenomas. OBJECTIVE: To identify genetic abnormalities in nonhereditary parathyroid adenomas by whole-exome sequence analysis. DESIGN: Whole-exome sequence analysis was performed on parathyroid adenomas and leukocyte DNA samples from 16 postmenopausal women without a family history of parathyroid tumors or MEN1 and in whom primary hyperparathyroidism due to single-gland disease was cured by surgery. Somatic variants confirmed in this discovery set were assessed in 24 other parathyroid adenomas. RESULTS: Over 90% of targeted exons were captured and represented by more than 10 base reads. Analysis identified 212 somatic variants (median eight per tumor; range, 2-110), with the majority being heterozygous nonsynonymous single-nucleotide variants that predicted missense amino acid substitutions. Somatic MEN1 mutations occurred in six of 16 (∼35%) parathyroid adenomas, in association with loss of heterozygosity on chromosome 11. However, no other gene was mutated in more than one tumor. Mutations in several genes that may represent low-frequency driver mutations were identified, including a protection of telomeres 1 (POT1) mutation that resulted in exon skipping and disruption to the single-stranded DNA-binding domain, which may contribute to increased genomic instability and the observed high mutation rate in one tumor. CONCLUSIONS: Parathyroid adenomas typically harbor few somatic variants, consistent with their low proliferation rates. MEN1 mutation represents the major driver in sporadic parathyroid tumorigenesis although multiple low-frequency driver mutations likely account for tumors not harboring somatic MEN1 mutations.