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Pubertal timing varies considerably and is associated with later health outcomes. We performed multi-ancestry genetic analyses on ~800,000 women, identifying 1,080 signals for age at menarche. Collectively, these explained 11% of trait variance in an independent sample. Women at the top and bottom 1% of polygenic risk exhibited ~11 and ~14-fold higher risks of delayed and precocious puberty, respectively. We identified several genes harboring rare loss-of-function variants in ~200,000 women, including variants in ZNF483, which abolished the impact of polygenic risk. Variant-to-gene mapping approaches and mouse gonadotropin-releasing hormone neuron RNA sequencing implicated 665 genes, including an uncharacterized G-protein-coupled receptor, GPR83, which amplified the signaling of MC3R, a key nutritional sensor. Shared signals with menopause timing at genes involved in DNA damage response suggest that the ovarian reserve might signal centrally to trigger puberty. We also highlight body size-dependent and independent mechanisms that potentially link reproductive timing to later life disease.

Birth weight variation is influenced by fetal and maternal genetic and non-genetic factors, and has been reproducibly associated with future cardio-metabolic health outcomes. In expanded genome-wide association analyses of own birth weight (n = 321,223) and offspring birth weight (n = 230,069 mothers), we identified 190 independent association signals (129 of which are novel). We used structural equation modeling to decompose the contributions of direct fetal and indirect maternal genetic effects, then applied Mendelian randomization to illuminate causal pathways. For example, both indirect maternal and direct fetal genetic effects drive the observational relationship between lower birth weight and higher later blood pressure: maternal blood pressure-raising alleles reduce offspring birth weight, but only direct fetal effects of these alleles, once inherited, increase later offspring blood pressure. Using maternal birth weight-lowering genotypes to proxy for an adverse intrauterine environment provided no evidence that it causally raises offspring blood pressure, indicating that the inverse birth weight-blood pressure association is attributable to genetic effects, and not to intrauterine programming.

Whole exome sequencing (WES), targeted gene panel sequencing and single nucleotide polymorphism (SNP) arrays are increasingly used for the identification of actionable alterations that are critical to cancer care. Here, we compared The Cancer Genome Atlas (TCGA) and the Genomics Evidence Neoplasia Information Exchange (GENIE) breast cancer genomic datasets (array and next generation sequencing (NGS) data) in detecting genomic alterations in clinically relevant genes. We performed an in silico analysis to determine the concordance in the frequencies of actionable mutations and copy number alterations/aberrations (CNAs) in the two most common breast cancer histologies, invasive lobular and invasive ductal carcinoma. We found that targeted sequencing identified a larger number of mutational hotspots and clinically significant amplifications that would have been missed by WES and SNP arrays in many actionable genes such as PIK3CA, EGFR, AKT3, FGFR1, ERBB2, ERBB3 and ESR1. The striking differences between the number of mutational hotspots and CNAs generated from these platforms highlight a number of factors that should be considered in the interpretation of array and NGS-based genomic data for precision medicine. Targeted panel sequencing was preferable to WES to define the full spectrum of somatic mutations present in a tumor.

BackgroundWe characterized the whole transcriptome of circulating tumor cells (CTCs) in stage II-III breast cancer to evaluate correlations with primary tumor biology.MethodsCTCs were isolated from peripheral blood (PB) via immunomagnetic enrichment followed by fluorescence-activated cell sorting (IE/FACS). CTCs, PB, and fresh tumors were profiled using RNA-seq. Formalin-fixed, paraffin-embedded (FFPE) tumors were subjected to RNA-seq and NanoString PAM50 assays with risk of recurrence (ROR) scores.ResultsCTCs were detected in 29/33 (88%) patients. We selected 21 cases to attempt RNA-seq (median number of CTCs = 9). Sixteen CTC samples yielded results that passed quality-control metrics, and these samples had a median of 4,311,255 uniquely mapped reads (less than PB or tumors). Intrinsic subtype predicted by comparing estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) versus PAM50 for FFPE tumors was 85% concordant. However, CTC RNA-seq subtype assessed by the PAM50 classification genes was highly discordant, both with the subtype predicted by ER/PR/HER2 and by PAM50 tumors. Two patients died of metastatic disease, both of whom had high ROR scores and high CTC counts. We identified significant genes, canonical pathways, upstream regulators, and molecular interaction networks comparing CTCs by various clinical factors. We also identified a 75-gene signature with highest expression in CTCs and tumors taken together that was prognostic in The Cancer Genome Atlas and Molecular Taxonomy of Breast Cancer International Consortium datasets.ConclusionIt is feasible to use RNA-seq of CTCs in non-metastatic patients to discover novel tumor biology characteristics.

Although next-generation sequencing has revolutionized the ability to associate variants with human diseases, diagnostic rates and development of new therapies are still limited by a lack of knowledge of the functions and pathobiological mechanisms of most genes. To address this challenge, the International Mouse Phenotyping Consortium is creating a genome- and phenome-wide catalog of gene function by characterizing new knockout-mouse strains across diverse biological systems through a broad set of standardized phenotyping tests. All mice will be readily available to the biomedical community. Analyzing the first 3,328 genes identified models for 360 diseases, including the first models, to our knowledge, for type C Bernard-Soulier, Bardet-Biedl-5 and Gordon Holmes syndromes. 90% of our phenotype annotations were novel, providing functional evidence for 1,092 genes and candidates in genetically uncharacterized diseases including arrhythmogenic right ventricular dysplasia 3. Finally, we describe our role in variant functional validation with The 100,000 Genomes Project and others.

Programmed cell death protein 1 (PD-1) is an immune checkpoint receptor that is upregulated on activated T cells for the induction of immune tolerance. Tumour cells frequently overexpress the ligand for PD-1, programmed cell death ligand 1 (PD-L1), facilitating their escape from the immune system. Monoclonal antibodies that block the interaction between PD-1 and PD-L1, by binding to either the ligand or receptor, have shown notable clinical efficacy in patients with a variety of cancers, including melanoma, colorectal cancer, non-small-cell lung cancer and Hodgkin's lymphoma. Although it is well established that PD-1-PD-L1 blockade activates T cells, little is known about the role that this pathway may have in tumour-associated macrophages (TAMs). Here we show that both mouse and human TAMs express PD-1. TAM PD-1 expression increases over time in mouse models of cancer and with increasing disease stage in primary human cancers. TAM PD-1 expression correlates negatively with phagocytic potency against tumour cells, and blockade of PD-1-PD-L1 in vivo increases macrophage phagocytosis, reduces tumour growth and lengthens the survival of mice in mouse models of cancer in a macrophage-dependent fashion. This suggests that PD-1-PD-L1 therapies may also function through a direct effect on macrophages, with substantial implications for the treatment of cancer with these agents.

Birth weight (BW) has been shown to be influenced by both fetal and maternal factors and in observational studies is reproducibly associated with future risk of adult metabolic diseases including type 2 diabetes (T2D) and cardiovascular disease. These life-course associations have often been attributed to the impact of an adverse early life environment. Here, we performed a multi-ancestry genome-wide association study (GWAS) meta-analysis of BW in 153,781 individuals, identifying 60 loci where fetal genotype was associated with BW (P

PurposeThe potential utility of circulating tumor cells (CTCs) as liquid biopsies is of great interest. We hypothesized that CTC capture using EpCAM based gating is feasible for most breast cancer subtypes.ResultsCancer cells could be recovered from all intrinsic subtypes of breast cancer with IE/FACS, however, claudin-low cell lines showed very low capture rates compared to the four other groups (p = 0.03). IE/FACS detection of CTC mimic cells was time sensitive, emphasizing controlling for pre-analytic variables in CTC studies. Median fluorescent intensity for flow cytometry and RNA flow cell type characterization were highly correlated, predicting for CTC isolation across molecular subtypes. RNA-Seq of IE/FACS sorted single cell equivalents showed high correlation compared to bulk cell lines, and distinct gene expression signatures compared to PB.Materials and methodsTen cell lines representing all major subtypes of breast cancer were spiked (as CTC mimics) into and recovered from peripheral blood (PB) using immunomagnetic enrichment followed by fluorescence-activated cell sorting (IE/FACS). Flow cytometry and RNA flow were used to quantify the expression of multiple breast cancer related markers of interest. Two different RNA-Seq technologies were used to analyze global gene expression of recovered sorted cells compared to bulk cell lines and PB.ConclusionsEpCAM based IE/FACS detected and captured a portion of spiked cells from each of the 10 cell lines representing all breast cancer subtypes, including basal-like but not claudin-low cancers. The assay allows for the isolation of high quality RNA suitable for accurate RNA-Seq of heterogeneous rare cell populations.

The extent to which low-frequency (minor allele frequency (MAF) between 1-5%) and rare (MAF ≤ 1%) variants contribute to complex traits and disease in the general population is mainly unknown. Bone mineral density (BMD) is highly heritable, a major predictor of osteoporotic fractures, and has been previously associated with common genetic variants, as well as rare, population-specific, coding variants. Here we identify novel non-coding genetic variants with large effects on BMD (ntotal = 53,236) and fracture (ntotal = 508,253) in individuals of European ancestry from the general population. Associations for BMD were derived from whole-genome sequencing (n = 2,882 from UK10K (ref. 10); a population-based genome sequencing consortium), whole-exome sequencing (n = 3,549), deep imputation of genotyped samples using a combined UK10K/1000 Genomes reference panel (n = 26,534), and de novo replication genotyping (n = 20,271). We identified a low-frequency non-coding variant near a novel locus, EN1, with an effect size fourfold larger than the mean of previously reported common variants for lumbar spine BMD (rs11692564(T), MAF = 1.6%, replication effect size = +0.20 s.d., Pmeta = 2 × 10(-14)), which was also associated with a decreased risk of fracture (odds ratio = 0.85; P = 2 × 10(-11); ncases = 98,742 and ncontrols = 409,511). Using an En1(cre/flox) mouse model, we observed that conditional loss of En1 results in low bone mass, probably as a consequence of high bone turnover. We also identified a novel low-frequency non-coding variant with large effects on BMD near WNT16 (rs148771817(T), MAF = 1.2%, replication effect size = +0.41 s.d., Pmeta = 1 × 10(-11)). In general, there was an excess of association signals arising from deleterious coding and conserved non-coding variants. These findings provide evidence that low-frequency non-coding variants have large effects on BMD and fracture, thereby providing rationale for whole-genome sequencing and improved imputation reference panels to study the genetic architecture of complex traits and disease in the general population.

Age-related impairment of neuromuscular activation has been shown to contribute to weakness in older adults. However, it is unclear to what extent impaired neuromuscular activation independently accounts for decline of mobility function. The hypothesis of this study is that the capability to produce rapid neuromuscular activation during maximal effort leg muscle contractions will be shown to be an independent predictor of mobility function in older men and women after accounting for muscle size and adiposity, body composition and age. Twenty six older men and eighteen older women (aged 70-85years) participated in this study. Mobility function was assessed by the 400-m walk test. Neuromuscular activation of the quadriceps muscle group was assessed by surface electromyography ("rate of EMG rise"). Thigh muscle cross sectional area and adiposity were assessed by computed tomography. In males, univariate regression analysis revealed strong associations between walking speed and a number of predictors including age (p

BackgroundIt is assumed that the oxytocin receptor gene (OXTR) is associated with factors that are related to features of reproduction as well as the currently emerging fields of mood and emotional response.MethodsWe analysed data from over 8000 mothers who participated in the Avon Longitudinal Study of Parents and Children (ALSPAC). We determined reproductive, emotional and personality differences related to the two SNPs rs53576 and rs2254298 of the oxytocin receptor gene to determine whether there was evidence in this population for: (i) associations with emotional and personality differences, and (ii) behavioural or environmental links with these SNPs using a hypothesis free approach with over 1000 types of exposure.ResultsOur analyses of 7723 women showed that there were no differences in 11 mood, social or relationship characteristics associated with the rs2254298, and just one with rs53576 (with emotional loneliness)--one statistically significant out of 22 tests is no more than would be expected by chance. There were no interactions with childhood abuse. Using a hypothesis-free approach we found few indicators of environmental or behavioural differences associated with rs2254298, but there was an excess of associations with eating habits with rs53576. The findings included an association with dieting to lose weight, and habits typical of bulimia for the women with GG. The nutrition of the women also showed negative associations of the GG genotype with 13 nutrients, including vitamins D, B12 and retinol, and intake of calcium, potassium and iodine.ConclusionsWe conclude that this large database of pregnant women was unable to provide confirmation of the types of personality associated with these two OXTR SNPs, but we have shown some evidence of eating differences in those with GG on rs53576. Confirmation of our hypothesis free associations using other data sets is important.

Written and verbal languages are neurobehavioral traits vital to the development of communication skills. Unfortunately, disorders involving these traits-specifically reading disability (RD) and language impairment (LI)-are common and prevent affected individuals from developing adequate communication skills, leaving them at risk for adverse academic, socioeconomic and psychiatric outcomes. Both RD and LI are complex traits that frequently co-occur, leading us to hypothesize that these disorders share genetic etiologies. To test this, we performed a genome-wide association study on individuals affected with both RD and LI in the Avon Longitudinal Study of Parents and Children. The strongest associations were seen with markers in ZNF385D (OR = 1.81, P = 5.45 × 10(-7) ) and COL4A2 (OR = 1.71, P = 7.59 × 10(-7) ). Markers within NDST4 showed the strongest associations with LI individually (OR = 1.827, P = 1.40 × 10(-7) ). We replicated association of ZNF385D using receptive vocabulary measures in the Pediatric Imaging Neurocognitive Genetics study (P = 0.00245). We then used diffusion tensor imaging fiber tract volume data on 16 fiber tracts to examine the implications of replicated markers. ZNF385D was a predictor of overall fiber tract volumes in both hemispheres, as well as global brain volume. Here, we present evidence for ZNF385D as a candidate gene for RD and LI. The implication of transcription factor ZNF385D in RD and LI underscores the importance of transcriptional regulation in the development of higher order neurocognitive traits. Further study is necessary to discern target genes of ZNF385D and how it functions within neural development of fluent language.

Refractive errors are common eye disorders of public health importance worldwide. Ocular axial length (AL) is the major determinant of refraction and thus of myopia and hyperopia. We conducted a meta-analysis of genome-wide association studies for AL, combining 12,531 Europeans and 8,216 Asians. We identified eight genome-wide significant loci for AL (RSPO1, C3orf26, LAMA2, GJD2, ZNRF3, CD55, MIP, and ALPPL2) and confirmed one previously reported AL locus (ZC3H11B). Of the nine loci, five (LAMA2, GJD2, CD55, ALPPL2, and ZC3H11B) were associated with refraction in 18 independent cohorts (n = 23,591). Differential gene expression was observed for these loci in minus-lens-induced myopia mouse experiments and human ocular tissues. Two of the AL genes, RSPO1 and ZNRF3, are involved in Wnt signaling, a pathway playing a major role in the regulation of eyeball size. This study provides evidence of shared genes between AL and refraction, but importantly also suggests that these traits may have unique pathways.

Carbon dioxide (CO(2)) present in exhaled air is the most important sensory cue for female blood-feeding mosquitoes, causing activation of long-distance host-seeking flight, navigation towards the vertebrate host and, in the case of Aedes aegypti, increased sensitivity to skin odours. The CO(2) detection machinery is therefore an ideal target to disrupt host seeking. Here we use electrophysiological assays to identify a volatile odorant that causes an unusual, ultra-prolonged activation of CO(2)-detecting neurons in three major disease-transmitting mosquitoes: Anopheles gambiae, Culex quinquefasciatus and A. aegypti. Importantly, ultra-prolonged activation of these neurons severely compromises their ability subsequently to detect CO(2) for several minutes. We also identify odours that strongly inhibit CO(2)-sensitive neurons as candidates for use in disruption of host-seeking behaviour, as well as an odour that evokes CO(2)-like activity and thus has potential use as a lure in trapping devices. Analysis of responses to panels of structurally related odours across the three mosquitoes and Drosophila, which have related CO(2)-receptor proteins, reveals a pattern of inhibition that is often conserved. We use video tracking in wind-tunnel experiments to demonstrate that the novel ultra-prolonged activators can completely disrupt CO(2)-mediated activation as well as source-finding behaviour in Aedes mosquitoes, even after the odour is no longer present. Lastly, semi-field studies demonstrate that use of ultra-prolonged activators disrupts CO(2)-mediated hut entry behaviour of Culex mosquitoes. The three classes of CO(2)-response-modifying odours offer powerful instruments for developing new generations of insect repellents and lures, which even in small quantities can interfere with the ability of mosquitoes to seek humans.

Trains of 20-ms-duration pulses of pheromone were delivered at rates of 1-33 Hz to antennal preparations of males of Bombyx mori and Lymantria dispar, two moth species with bipectinate antennae. Resolution of rapidly pulsed plumes of pheromone was not compromised by a complex antennal morphology or by moderate changes in wind speed (25-50 cm/s). Fourier analysis of the electroantennograms resolved the temporal structure of the signal at frequencies up to 25 Hz for B. mori and up to 5 Hz for L. dispar. The ability of these sensory structures to identify the original (unchanged) frequency of the pulse train is particularly noteworthy because air is slowed by about an order of magnitude as it passes through bipectinate antennae. Although an unchanging frequency in slowed airflow may be counterintuitive, this flow pattern, and its effects on odorant patch shape and spacing, is explained from fluid mechanical principles (i.e., the principle of continuity). An unchanging frequency suggests that as decelerating air passes through a bipectinate antenna, the slowed patches of odorant are stretched, thinned, and brought closer together by the same factor with which they are slowed.

This article describes the ongoing collaborative effort of six research teams to operationalize and execute an integrative approach to the study of gene x environment interactions in the development of tobacco dependence. At the core of the project is a longitudinal investigation of social and behavioral risk factors for tobacco use in individuals who were, on average, 13 years of age at intake and for whom smoking outcomes extending from early adolescence to young adulthood have been characterized previously (current average age of the cohort is 29 years). The conceptual framework for the integrative approach and the longitudinal investigation on which the study is based is presented. A description is also provided of the methods used to: (a) recruit participants and families to provide DNA samples and information on tobacco use; (b) assess participants for relevant tobacco-related phenotypes including smoking history, current use of tobacco, and nicotine metabolism; (c) assess the quality of the DNA samples collected from participants for genome-wide scanning and candidate gene analysis; (d) examine several research questions concerning the role of genetic and environmental factors in the onset and maintenance of tobacco use; and (e) ensure adherence to local and federal guidelines for ethical and legal investigations of genotypic associations with tobacco-related phenotypes in families. This investigation is unique among ongoing studies of the genetics of tobacco dependence in the extent to which equal importance has been assigned to both phenotypic and genotypic measurements.

Abstract Background No study to date has looked at the gender of emergency medicine (EM) physicians in the United States in relation to admission rates. This study seeks to investigate admission rates of adult patients treated by female vs male EM physicians, to identify whether a practice pattern bias exists. Methods This was a multicenter retrospective study of four community hospitals. Population: All patient encounters between July 1, 2016 and June 30, 2017. Outcome: We compared multiple benchmarks, including admission rates, patient acuity, length of stay, return visits, patient age, and years of practice using descriptive statistics and Pearson Correlation Coefficients. Results 171,762 encounters by 71 EM physicians; 29 females, 42 males. Average admission rates: female 30.1%, male 28.0%, p = .188. Average encounters: female 2456, male 2394, p = 0.77. Acuity: female 149.3, male 146.9, p = .227. Average length of stay (minutes): female 294.4, male 277.4, p = .137. Average patient age: female 50.9, male 50.2, p = .457. Median time of encounter: female 12.8, male 12.7, p = .964. Years of practice: female 16.2, male 19.1, p = .274. Average return visits per one thousand: female 8.5, male 8.5, p = .864. Secondary analysis of Pearson Correlation Coefficient of Significance; admission rate and length of stay: female 0.53, p = .0026; male 0.76, p