Amplification-free detection of SARS-CoV-2 with CRISPR-Cas13a and mobile phone microscopy.

The December 2019 outbreak of a novel respiratory virus, SARS-CoV-2, has become an ongoing global pandemic due in part to the challenge of identifying symptomatic, asymptomatic, and pre-symptomatic carriers of the virus. CRISPR diagnostics can augment gold-standard PCR-based testing if they can be made rapid, portable, and accurate. Here, we report the development of an amplification-free CRISPR-Cas13a assay for direct detection of SARS-CoV-2 from nasal swab RNA that can be read with a mobile phone microscope. The assay achieved ∼100 copies/µL sensitivity in under 30 min of measurement time and accurately detected pre-extracted RNA from a set of positive clinical samples in under 5 min. We combined crRNAs targeting SARS-CoV-2 RNA to improve sensitivity and specificity and directly quantified viral load using enzyme kinetics. Integrated with a reader device based on a mobile phone, this assay has the potential to enable rapid, low-cost, point-of-care screening for SARS-CoV-2.

Caspase substrates and cellular remodeling.

The caspases are unique proteases that mediate the major morphological changes of apoptosis and various other cellular remodeling processes. As we catalog and study the myriad proteins subject to cleavage by caspases, we are beginning to appreciate the full functional repertoire of these enzymes. Here, we examine current knowledge about caspase cleavages: what kinds of proteins are cut, in what contexts, and to what end. After reviewing basic caspase biology, we describe the technologies that enable high-throughput caspase substrate discovery and the datasets they have yielded. We discuss how caspases recognize their substrates and how cleavages are conserved among different metazoan organisms. Rather than comprehensively reviewing all known substrates, we use examples to highlight some functional impacts of caspase cuts during apoptosis and differentiation. Finally, we discuss the roles caspase substrates can play in medicine. Though great progress has been made in this field, many important areas still await exploration.

Dynamic Regulation of Long-Chain Fatty Acid Oxidation by a Noncanonical Interaction between the MCL-1 BH3 Helix and VLCAD.

MCL-1 is a BCL-2 family protein implicated in the development and chemoresistance of human cancer. Unlike its anti-apoptotic homologs, Mcl-1 deletion has profound physiologic consequences, indicative of a broader role in homeostasis. We report that the BCL-2 homology 3 (BH3) α helix of MCL-1 can directly engage very long-chain acyl-CoA dehydrogenase (VLCAD), a key enzyme of the mitochondrial fatty acid ß-oxidation (FAO) pathway. Proteomic analysis confirmed that the mitochondrial matrix isoform of MCL-1 (MCL-1Matrix) interacts with VLCAD. Mcl-1 deletion, or eliminating MCL-1Matrix alone, selectively deregulated long-chain FAO, causing increased flux through the pathway in response to nutrient deprivation. Transient elevation in MCL-1 upon serum withdrawal, a striking increase in MCL-1 BH3/VLCAD interaction upon palmitic acid titration, and direct modulation of enzymatic activity by the MCL-1 BH3 α helix are consistent with dynamic regulation. Thus, the MCL-1 BH3 interaction with VLCAD revealed a separable, gain-of-function role for MCL-1 in the regulation of lipid metabolism.

FLASH-TB: an Application of Next-Generation CRISPR to Detect Drug Resistant Tuberculosis from Direct Sputum.

Offering patients with tuberculosis (TB) an optimal and timely treatment regimen depends on the rapid detection of Mycobacterium tuberculosis (Mtb) drug resistance from clinical samples. Finding Low Abundance Sequences by Hybridization (FLASH) is a technique that harnesses the efficiency, specificity, and flexibility of the Cas9 enzyme to enrich targeted sequences. Here, we used FLASH to amplify 52 candidate genes probably associated with resistance to first- and second-line drugs in the Mtb reference strain (H37Rv), then detect drug resistance mutations in cultured Mtb isolates, and in sputum samples. 92% of H37Rv reads mapped to Mtb targets, with 97.8% of target regions covered at a depth ≥ 10X. Among cultured isolates, FLASH-TB detected the same 17 drug resistance mutations as whole genome sequencing (WGS) did, but with much greater depth. Among the 16 sputum samples, FLASH-TB increased recovery of Mtb DNA compared with WGS (from 1.4% [IQR 0.5-7.5] to 33% [IQR 4.6-66.3]) and average depth reads of targets (from 6.3 [IQR 3.8-10.5] to 1991 [IQR 254.4-3623.7]). FLASH-TB identified Mtb complex in all 16 samples based on IS1081 and IS6110 copies. Drug resistance predictions for 15/16 (93.7%) clinical samples were highly concordant with phenotypic DST for isoniazid, rifampicin, amikacin, and kanamycin [15/15 (100%)], ethambutol [12/15 (80%)] and moxifloxacin [14/15 (93.3%)]. These results highlighted the potential of FLASH-TB for detecting Mtb drug resistance from sputum samples.

A compact Cascade-Cas3 system for targeted genome engineering.

CRISPR-Cas technologies have enabled programmable gene editing in eukaryotes and prokaryotes. However, the leading Cas9 and Cas12a enzymes are limited in their ability to make large deletions. Here, we used the processive nuclease Cas3, together with a minimal Type I-C Cascade-based system for targeted genome engineering in bacteria. DNA cleavage guided by a single CRISPR RNA generated large deletions (7-424 kilobases) in Pseudomonas aeruginosa with near-100% efficiency, while Cas9 yielded small deletions and point mutations. Cas3 generated bidirectional deletions originating from the programmed site, which was exploited to reduce the P. aeruginosa genome by 837 kb (13.5%). Large deletion boundaries were efficiently specified by a homology-directed repair template during editing with Cascade-Cas3, but not Cas9. A transferable 'all-in-one' vector was functional in Escherichia coli, Pseudomonas syringae and Klebsiella pneumoniae, and endogenous CRISPR-Cas use was enhanced with an 'anti-anti-CRISPR' strategy. P. aeruginosa Type I-C Cascade-Cas3 (PaeCas3c) facilitates rapid strain manipulation with applications in synthetic biology, genome minimization and the removal of large genomic regions.

Sensitive, Highly Multiplexed Sequencing of Microhaplotypes From the Plasmodium falciparum Heterozygome.

BACKGROUND: Targeted next-generation sequencing offers the potential for consistent, deep coverage of information-rich genomic regions to characterize polyclonal Plasmodium falciparum infections. However, methods to identify and sequence these genomic regions are currently limited. METHODS: A bioinformatic pipeline and multiplex methods were developed to identify and simultaneously sequence 100 targets and applied to dried blood spot (DBS) controls and field isolates from Mozambique. For comparison, whole-genome sequencing data were generated for the same controls. RESULTS: Using publicly available genomes, 4465 high-diversity genomic regions suited for targeted sequencing were identified, representing the P. falciparum heterozygome. For this study, 93 microhaplotypes with high diversity (median expected heterozygosity = 0.7) were selected along with 7 drug resistance loci. The sequencing method achieved very high coverage (median 99%), specificity (99.8%), and sensitivity (90% for haplotypes with 5% within sample frequency in dried blood spots with 100 parasites/µL). In silico analyses revealed that microhaplotypes provided much higher resolution to discriminate related from unrelated polyclonal infections than biallelic single-nucleotide polymorphism barcodes. CONCLUSIONS: The bioinformatic and laboratory methods outlined here provide a flexible tool for efficient, low-cost, high-throughput interrogation of the P. falciparum genome, and can be tailored to simultaneously address multiple questions of interest in various epidemiological settings.

Performance Characteristics of a Rapid Severe Acute Respiratory Syndrome Coronavirus 2 Antigen Detection Assay at a Public Plaza Testing Site in San Francisco.

We evaluated the performance of the Abbott BinaxNOW rapid antigen test for coronavirus disease 2019 (Binax-CoV2) to detect virus among persons, regardless of symptoms, at a public plaza site of ongoing community transmission. Titration with cultured severe acute respiratory syndrome coronavirus 2 yielded a human observable threshold between 1.6 × 104-4.3 × 104 viral RNA copies (cycle threshold [Ct], 30.3-28.8). Among 878 subjects tested, 3% (26 of 878) were positive by reverse-transcription polymerase chain reaction, of whom 15 of 26 had a Ct <30, indicating high viral load; of these, 40% (6 of 15) were asymptomatic. Using this Ct threshold (<30) for Binax-CoV2 evaluation, the sensitivity of Binax-CoV2 was 93.3% (95% confidence interval, 68.1%-99.8%) (14 of 15) and the specificity was 99.9% (99.4%-99.9%) (855 of 856).

Community Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 Disproportionately Affects the Latinx Population During Shelter-in-Place in San Francisco.

BACKGROUND: There is an urgent need to understand the dynamics and risk factors driving ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission during shelter-in-place mandates. METHODS: We offered SARS-CoV-2 reverse-transcription polymerase chain reaction (PCR) and antibody (Abbott ARCHITECT IgG) testing, regardless of symptoms, to all residents (aged ≥4 years) and workers in a San Francisco census tract (population: 5174) at outdoor, community-mobilized events over 4 days. We estimated SARS-CoV-2 point prevalence (PCR positive) and cumulative incidence (antibody or PCR positive) in the census tract and evaluated risk factors for recent (PCR positive/antibody negative) vs prior infection (antibody positive/PCR negative). SARS-CoV-2 genome recovery and phylogenetics were used to measure viral strain diversity, establish viral lineages present, and estimate number of introductions. RESULTS: We tested 3953 persons (40% Latinx; 41% White; 9% Asian/Pacific Islander; and 2% Black). Overall, 2.1% (83/3871) tested PCR positive: 95% were Latinx and 52% were asymptomatic when tested; 1.7% of census tract residents and 6.0% of workers (non-census tract residents) were PCR positive. Among 2598 tract residents, estimated point prevalence of PCR positives was 2.3% (95% confidence interval [CI], 1.2%-3.8%): 3.9% (95% CI, 2.0%-6.4%) among Latinx persons vs 0.2% (95% CI, .0-.4%) among non-Latinx persons. Estimated cumulative incidence among residents was 6.1% (95% CI, 4.0%-8.6%). Prior infections were 67% Latinx, 16% White, and 17% other ethnicities. Among recent infections, 96% were Latinx. Risk factors for recent infection were Latinx ethnicity, inability to shelter in place and maintain income, frontline service work, unemployment, and household income <$50 000/year. Five SARS-CoV-2 phylogenetic lineages were detected. CONCLUSIONS: SARS-CoV-2 infections from diverse lineages continued circulating among low-income, Latinx persons unable to work from home and maintain income during San Francisco's shelter-in-place ordinance.

FLASH: a next-generation CRISPR diagnostic for multiplexed detection of antimicrobial resistance sequences.

The growing prevalence of deadly microbes with resistance to previously life-saving drug therapies is a dire threat to human health. Detection of low abundance pathogen sequences remains a challenge for metagenomic Next Generation Sequencing (NGS). We introduce FLASH (Finding Low Abundance Sequences by Hybridization), a next-generation CRISPR/Cas9 diagnostic method that takes advantage of the efficiency, specificity and flexibility of Cas9 to enrich for a programmed set of sequences. FLASH-NGS achieves up to 5 orders of magnitude of enrichment and sub-attomolar gene detection with minimal background. We provide an open-source software tool (FLASHit) for guide RNA design. Here we applied it to detection of antimicrobial resistance genes in respiratory fluid and dried blood spots, but FLASH-NGS is applicable to all areas that rely on multiplex PCR.

Integrating host response and unbiased microbe detection for lower respiratory tract infection diagnosis in critically ill adults.

Lower respiratory tract infections (LRTIs) lead to more deaths each year than any other infectious disease category. Despite this, etiologic LRTI pathogens are infrequently identified due to limitations of existing microbiologic tests. In critically ill patients, noninfectious inflammatory syndromes resembling LRTIs further complicate diagnosis. To address the need for improved LRTI diagnostics, we performed metagenomic next-generation sequencing (mNGS) on tracheal aspirates from 92 adults with acute respiratory failure and simultaneously assessed pathogens, the airway microbiome, and the host transcriptome. To differentiate pathogens from respiratory commensals, we developed a rules-based model (RBM) and logistic regression model (LRM) in a derivation cohort of 20 patients with LRTIs or noninfectious acute respiratory illnesses. When tested in an independent validation cohort of 24 patients, both models achieved accuracies of 95.5%. We next developed pathogen, microbiome diversity, and host gene expression metrics to identify LRTI-positive patients and differentiate them from critically ill controls with noninfectious acute respiratory illnesses. When tested in the validation cohort, the pathogen metric performed with an area under the receiver-operating curve (AUC) of 0.96 (95% CI, 0.86-1.00), the diversity metric with an AUC of 0.80 (95% CI, 0.63-0.98), and the host transcriptional classifier with an AUC of 0.88 (95% CI, 0.75-1.00). Combining these achieved a negative predictive value of 100%. This study suggests that a single streamlined protocol offering an integrated genomic portrait of pathogen, microbiome, and host transcriptome may hold promise as a tool for LRTI diagnosis.

Modern contraceptive use among unmarried girls aged 15-19 years in South Western Nigeria: results from a cross-sectional baseline survey for the Adolescent 360 (A360) impact evaluation.

PURPOSE: Adolescents 360 (A360) is an initiative being rolled out across Nigeria with the aim of increasing voluntary modern contraception use among women aged 15 to 19 years. Using evaluation study baseline data, we identified sexuality, fertility and contraceptive use characteristics of young unmarried girls in South Western Nigeria. METHODS: A cross-sectional baseline survey of unmarried girls aged 15 to 19 years was conducted in Ogun state, Nigeria in August 2017. A clustered sampling design was used. We identified determinants of modern contraceptive use in this subpopulation using logistic regression. RESULTS: Of 12,024 women interviewed, 15.3% reported sexual intercourse in the past year. The majority of respondents (79.6%, 9525/11,967) had heard of contraception. 45.3% of sexually active respondents were using a modern contraceptive method. Of those using any method of contraception, male condoms (50.3%) were the most widely used modern method followed by the emergency contraceptive pill (16.7%). Following adjustment for socio-demographic characteristics, there was evidence that the use of modern contraception was positively associated with having never given birth, living in an urban area, current enrolment in education, high level of education, high socioeconomic status, exposure to information about contraception, perceived social support for contraception, and self-efficacy for contraception. CONCLUSIONS: In South Western Nigeria, unmarried sexually active adolescent girls have relatively low levels of modern contraceptive use. Programmes should aim to increase access to modern contraception and to increase social support and acceptability of contraceptive use.

Investigating Transfusion-related Sepsis Using Culture-Independent Metagenomic Sequencing.

BACKGROUND: Transfusion-related sepsis remains an important hospital infection control challenge. Investigation of septic transfusion events is often restricted by the limitations of bacterial culture in terms of time requirements and low yield in the setting of prior antibiotic administration. METHODS: In 3 gram-negative septic transfusion cases, we performed metagenomic next-generation sequencing (mNGS) of direct clinical blood specimens in addition to standard culture-based approaches utilized for infection control investigations. Pathogen detection leveraged IDSeq, a new open-access microbial bioinformatics portal. Phylogenetic analysis was performed to assess microbial genetic relatedness and understand transmission events. RESULTS: mNGS of direct clinical blood specimens afforded precision detection of pathogens responsible for each case of transfusion-related sepsis and enabled discovery of a novel Acinetobacter species in a platelet product that had become contaminated despite photochemical pathogen reduction. In each case, longitudinal assessment of pathogen burden elucidated the temporal sequence of events associated with each transfusion-transmitted infection. We found that informative data could be obtained from culture-independent mNGS of residual platelet products and leftover blood specimens that were either unsuitable or unavailable for culture or that failed to grow due to prior antibiotic administration. We additionally developed methods to enhance accuracy for detecting transfusion-associated pathogens that share taxonomic similarity to contaminants commonly found in mNGS library preparations. CONCLUSIONS: Culture-independent mNGS of blood products afforded rapid and precise assessment of pathogen identity, abundance, and genetic relatedness. Together, these challenging cases demonstrated the potential for metagenomics to advance existing methods for investigating transfusion-transmitted infections.

Sepsis from an apheresis platelet contaminated with Acinetobacter calcoaceticus/baumannii complex bacteria and Staphylococcus saprophyticus after pathogen reduction.

BACKGROUND: Strategies to reduce platelet (PLT) bacterial contamination include donor screening, skin disinfection, sample diversion, bacterial culture, pathogen reduction (PR), and day-of-transfusion tests. We report bacterial sepsis following a pathogen-reduced PLT transfusion. CASE REPORT: An adult male with relapsed acute lymphoblastic leukemia was successfully treated for central catheter-associated Staphylococcus aureus bacteremia. A peripherally inserted central catheter (PICC) was placed. Chills, rigors, and flushing developed immediately after PICC-infused pathogen-reduced PLTs, progressing to septic shock requiring intensive care management. METHODS: PICC and peripheral blood (PB), transfused bag saline flushes (TBFs), environmental samples, and the pathogen-reduced untransfused co-component (CC) were cultured. Plasma metagenomic and bacterial isolate whole-genome sequencing; PLT mitochondrial DNA (mtDNA) testing of untransfused CC and TBF; CC testing for amotosalen (S-59)/S-59 photoproducts; isolate PR studies (INTERCEPT); and TBF polymerase chain reaction for recipient Y-chromosome DNA were performed. RESULTS: PB and PICC cultures grew Acinetobacter calcoaceticus/baumannii complex (ACBC). TBF was gram-positive; mass spectrometry identified ACBC and Staphylococcus saprophyticus (SS). CC Gram stain and cultures were negative. Environmental cultures, some done after decontamination, were ACBC/SS negative. Posttransfusion patient plasma and TBF ACBC sequences were genetically identical. No Y-chromosome signal was detected in TBF. S-59 photoproducts and evidence of mtDNA amplification inhibition were found in the CC. Spiking PR studies showed >5.9-log inactivation for both isolates. Donor skin cultures for Acinetobacter were negative. CONCLUSION: CC sterility, PR studies, residual S-59 photoproducts, and mtDNA amplification inhibition suggest successful PR. Unidentified environmental sources and inherent or acquired bag defects may have contributed to postmanufacturing pathogen-reduced PLT contamination.

Synaptic, transcriptional and chromatin genes disrupted in autism.

The genetic architecture of autism spectrum disorder involves the interplay of common and rare variants and their impact on hundreds of genes. Using exome sequencing, here we show that analysis of rare coding variation in 3,871 autism cases and 9,937 ancestry-matched or parental controls implicates 22 autosomal genes at a false discovery rate (FDR) < 0.05, plus a set of 107 autosomal genes strongly enriched for those likely to affect risk (FDR < 0.30). These 107 genes, which show unusual evolutionary constraint against mutations, incur de novo loss-of-function mutations in over 5% of autistic subjects. Many of the genes implicated encode proteins for synaptic formation, transcriptional regulation and chromatin-remodelling pathways. These include voltage-gated ion channels regulating the propagation of action potentials, pacemaking and excitability-transcription coupling, as well as histone-modifying enzymes and chromatin remodellers-most prominently those that mediate post-translational lysine methylation/demethylation modifications of histones.

Modulating Pathogenesis with Mobile-CRISPRi.

Conditionally essential (CE) genes are required by pathogenic bacteria to establish and maintain infections. CE genes encode virulence factors, such as secretion systems and effector proteins, as well as biosynthetic enzymes that produce metabolites not found in the host environment. Due to their outsized importance in pathogenesis, CE gene products are attractive targets for the next generation of antimicrobials. However, the precise manipulation of CE gene expression in the context of infection is technically challenging, limiting our ability to understand the roles of CE genes in pathogenesis and accordingly design effective inhibitors. We previously developed a suite of CRISPR interference-based gene knockdown tools that are transferred by conjugation and stably integrate into bacterial genomes that we call Mobile-CRISPRi. Here, we show the efficacy of Mobile-CRISPRi in controlling CE gene expression in an animal infection model. We optimize Mobile-CRISPRi in Pseudomonas aeruginosa for use in a murine model of pneumonia by tuning the expression of CRISPRi components to avoid nonspecific toxicity. As a proof of principle, we demonstrate that knock down of a CE gene encoding the type III secretion system (T3SS) activator ExsA blocks effector protein secretion in culture and attenuates virulence in mice. We anticipate that Mobile-CRISPRi will be a valuable tool to probe the function of CE genes across many bacterial species and pathogenesis models.IMPORTANCE Antibiotic resistance is a growing threat to global health. To optimize the use of our existing antibiotics and identify new targets for future inhibitors, understanding the fundamental drivers of bacterial growth in the context of the host immune response is paramount. Historically, these genetic drivers have been difficult to manipulate precisely, as they are requisite for pathogen survival. Here, we provide the first application of Mobile-CRISPRi to study conditionally essential virulence genes in mouse models of lung infection through partial gene perturbation. We envision the use of Mobile-CRISPRi in future pathogenesis models and antibiotic target discovery efforts.

Pulmonary Metagenomic Sequencing Suggests Missed Infections in Immunocompromised Children.

BACKGROUND: Despite improved diagnostics, pulmonary pathogens in immunocompromised children frequently evade detection, leading to significant mortality. Therefore, we aimed to develop a highly sensitive metagenomic next-generation sequencing (mNGS) assay capable of evaluating the pulmonary microbiome and identifying diverse pathogens in the lungs of immunocompromised children. METHODS: We collected 41 lower respiratory specimens from 34 immunocompromised children undergoing evaluation for pulmonary disease at 3 children's hospitals from 2014-2016. Samples underwent mechanical homogenization, parallel RNA/DNA extraction, and metagenomic sequencing. Sequencing reads were aligned to the National Center for Biotechnology Information nucleotide reference database to determine taxonomic identities. Statistical outliers were determined based on abundance within each sample and relative to other samples in the cohort. RESULTS: We identified a rich cross-domain pulmonary microbiome that contained bacteria, fungi, RNA viruses, and DNA viruses in each patient. Potentially pathogenic bacteria were ubiquitous among samples but could be distinguished as possible causes of disease by parsing for outlier organisms. Samples with bacterial outliers had significantly depressed alpha-diversity (median, 0.61; interquartile range [IQR], 0.33-0.72 vs median, 0.96; IQR, 0.94-0.96; P < .001). Potential pathogens were detected in half of samples previously negative by clinical diagnostics, demonstrating increased sensitivity for missed pulmonary pathogens (P < .001). CONCLUSIONS: An optimized mNGS assay for pulmonary microbes demonstrates significant inoculation of the lower airways of immunocompromised children with diverse bacteria, fungi, and viruses. Potential pathogens can be identified based on absolute and relative abundance. Ongoing investigation is needed to determine the pathogenic significance of outlier microbes in the lungs of immunocompromised children with pulmonary disease.

Sexuality, fertility and family planning characteristics of married women aged 15 to 19 years in Ethiopia, Nigeria and Tanzania: a comparative analysis of cross-sectional data.

BACKGROUND: Adolescents 360 (A360) is an initiative being rolled out across Ethiopia, Nigeria and Tanzania with the aim of increasing uptake of voluntary modern contraception among sexually active women aged 15 to 19 years. Using evaluation baseline survey data, we described key sexuality, fertility and contraceptive use characteristics of married women aged 15 to 19 years living in three sub-national settings. METHODS: Cross-sectional baseline surveys of married women aged 15 to 19 years were conducted in Oromia (Ethiopia), Nasarawa (Northern Nigeria), and Mwanza (Tanzania) between August 2017 and February 2018. We also interviewed the husbands of a sub-group of married respondents to measure spousal acceptance and support for adolescent women to use modern contraception. A clustered sampling design was used in all three countries. We produced descriptive statistics on the socio-demographic and sexual and reproductive health characteristics of married women aged 15 to 19 years by study setting. RESULTS: In Oromia, Nasarawa and Mwanza, 31.4% (327/1198), 27.4% (1321/4816) and 7.5% (15/201) of married women surveyed had no education, and 68.3, 81.3 and 83.1% had ever been pregnant, respectively. Unmet need for modern contraception was 20.5, 21.9 and 32.0% in married women in Oromia, Nasarawa and Mwanza, made up almost entirely of unmet need for spacing. The vast majority of married women surveyed in Oromia (89.1%) and Mwanza (90.1%) had seen or heard about contraception in the last 12 months, compared to 30.1% of those surveyed in Nasarawa. Modern contraceptive prevalence (mCPR) was highest in married women aged 15 to 19 years in Oromia (47.2%), followed by Mwanza (19.4%) and Nasarawa (8.7%). Of those using a modern method of contraception in Oromia, 93.4% were using injectables or long-acting methods, compared to 49.4% in Nasarawa and 69.6% in Mwanza. CONCLUSIONS: Overall, unmet need for modern contraception is high among married women aged 15 to 19 years across the three settings. mCPR for married women aged 15 to 19 years is low in Nasarawa and Mwanza. Ultimately, no single intervention will suit all situations, but improving the quality, analyses and utilisation of subnational data can help decision-makers design more context specific interventions.

Patterns and rates of exonic de novo mutations in autism spectrum disorders.

Autism spectrum disorders (ASD) are believed to have genetic and environmental origins, yet in only a modest fraction of individuals can specific causes be identified. To identify further genetic risk factors, here we assess the role of de novo mutations in ASD by sequencing the exomes of ASD cases and their parents (n = 175 trios). Fewer than half of the cases (46.3%) carry a missense or nonsense de novo variant, and the overall rate of mutation is only modestly higher than the expected rate. In contrast, the proteins encoded by genes that harboured de novo missense or nonsense mutations showed a higher degree of connectivity among themselves and to previous ASD genes as indexed by protein-protein interaction screens. The small increase in the rate of de novo events, when taken together with the protein interaction results, are consistent with an important but limited role for de novo point mutations in ASD, similar to that documented for de novo copy number variants. Genetic models incorporating these data indicate that most of the observed de novo events are unconnected to ASD; those that do confer risk are distributed across many genes and are incompletely penetrant (that is, not necessarily sufficient for disease). Our results support polygenic models in which spontaneous coding mutations in any of a large number of genes increases risk by 5- to 20-fold. Despite the challenge posed by such models, results from de novo events and a large parallel case-control study provide strong evidence in favour of CHD8 and KATNAL2 as genuine autism risk factors.

Convergence of genes and cellular pathways dysregulated in autism spectrum disorders.

Rare copy-number variation (CNV) is an important source of risk for autism spectrum disorders (ASDs). We analyzed 2,446 ASD-affected families and confirmed an excess of genic deletions and duplications in affected versus control groups (1.41-fold, p = 1.0 × 10(-5)) and an increase in affected subjects carrying exonic pathogenic CNVs overlapping known loci associated with dominant or X-linked ASD and intellectual disability (odds ratio = 12.62, p = 2.7 × 10(-15), ∼3% of ASD subjects). Pathogenic CNVs, often showing variable expressivity, included rare de novo and inherited events at 36 loci, implicating ASD-associated genes (CHD2, HDAC4, and GDI1) previously linked to other neurodevelopmental disorders, as well as other genes such as SETD5, MIR137, and HDAC9. Consistent with hypothesized gender-specific modulators, females with ASD were more likely to have highly penetrant CNVs (p = 0.017) and were also overrepresented among subjects with fragile X syndrome protein targets (p = 0.02). Genes affected by de novo CNVs and/or loss-of-function single-nucleotide variants converged on networks related to neuronal signaling and development, synapse function, and chromatin regulation.

Genomic profiling of breast secretory carcinomas reveals distinct genetics from other breast cancers and similarity to mammary analog secretory carcinomas.

Secretory carcinomas of the breast are rare tumors with distinct histologic features, recurrent t(12;15)(p13;q25) translocation resulting in ETV6-NTRK3 gene fusion and indolent clinical behavior. Mammary analog secretory carcinomas arising in other sites are histopathologically similar to the breast tumors and also harbor ETV6-NTRK3 fusions. Breast secretory carcinomas are often triple (estrogen and progesterone receptor, HER2) negative with a basal-like immunophenotype. However, genomic studies are lacking, and whether these tumors share genetic features with other basal and/or triple negative breast cancers is unknown. Aside from shared ETV6-NTRK3 fusions, the genetic relatedness of secretory carcinomas arising in different sites is also uncertain. We immunoprofiled and sequenced 510 cancer-related genes in nine breast secretory carcinomas and six salivary gland mammary analog secretory carcinomas. Immunoprofiles of breast and salivary gland secretory carcinomas were similar. All the tumors showed strong diffuse MUC4 expression (n=15), and SOX10 was positive in all nine breast and in five out of six salivary gland tumors. All breast secretory carcinomas were triple negative or weakly ER-positive, and all tumors at both the sites expressed CK5/6 and/or EGFR, consistent with a basal-like phenotype. Sequencing revealed classic ETV6-NTRK3 fusion genes in all cases, including in carcinoma in situ of one breast tumor. Translocations were reciprocal and balanced in six out of nine breast and three out of six salivary gland tumors and were complex in three others. In contrast to most breast basal carcinomas, the mutational burden of secretory carcinomas was very low, and no additional pathogenic aberrations were identified in genes typically mutated in breast cancer. Five (56%) breast and two (33%) salivary gland tumors had simple genomes without copy number changes; the remainder had very few changes, averaging 1.3 per tumor. The ETV6-NTRK3 derivative chromosome was duplicated in one breast and one salivary gland tumor, and was the only copy number change in the latter. The findings highlight breast secretory carcinoma as a subtype more closely related to mammary analog secretory carcinoma than to basal/triple negative breast cancers of no special type. Lack of pathogenic mutations in common cancer-related genes suggests that ETV6-NTRK3 alone may suffice to drive these tumors and likely helps explain their indolent behavior.