Mapping the functional impact of non-coding regulatory elements in primary T cells through single-cell CRISPR screens.

BACKGROUND: Drug targets with genetic evidence are expected to increase clinical success by at least twofold. Yet, translating disease-associated genetic variants into functional knowledge remains a fundamental challenge of drug discovery. A key issue is that the vast majority of complex disease associations cannot be cleanly mapped to a gene. Immune disease-associated variants are enriched within regulatory elements found in T-cell-specific open chromatin regions. RESULTS: To identify genes and molecular programs modulated by these regulatory elements, we develop a CRISPRi-based single-cell functional screening approach in primary human T cells. Our pipeline enables the interrogation of transcriptomic changes induced by the perturbation of regulatory elements at scale. We first optimize an efficient CRISPRi protocol in primary CD4+ T cells via CROPseq vectors. Subsequently, we perform a screen targeting 45 non-coding regulatory elements and 35 transcription start sites and profile approximately 250,000 T -cell single-cell transcriptomes. We develop a bespoke analytical pipeline for element-to-gene (E2G) mapping and demonstrate that our method can identify both previously annotated and novel E2G links. Lastly, we integrate genetic association data for immune-related traits and demonstrate how our platform can aid in the identification of effector genes for GWAS loci. CONCLUSIONS: We describe "primary T cell crisprQTL" - a scalable, single-cell functional genomics approach for mapping regulatory elements to genes in primary human T cells. We show how this framework can facilitate the interrogation of immune disease GWAS hits and propose that the combination of experimental and QTL-based techniques is likely to address the variant-to-function problem.

fcfdr: an R package to leverage continuous and binary functional genomic data in GWAS.

BACKGROUND: Genome-wide association studies (GWAS) are limited in power to detect associations that exceed the stringent genome-wide significance threshold. This limitation can be alleviated by leveraging relevant auxiliary data, such as functional genomic data. Frameworks utilising the conditional false discovery rate have been developed for this purpose, and have been shown to increase power for GWAS discovery whilst controlling the false discovery rate. However, the methods are currently only applicable for continuous auxiliary data and cannot be used to leverage auxiliary data with a binary representation, such as whether SNPs are synonymous or non-synonymous, or whether they reside in regions of the genome with specific activity states. RESULTS: We describe an extension to the cFDR framework for binary auxiliary data, called "Binary cFDR". We demonstrate FDR control of our method using detailed simulations, and show that Binary cFDR performs better than a comparator method in terms of sensitivity and FDR control. We introduce an all-encompassing user-oriented CRAN R package ( https://annahutch.github.io/fcfdr/ ; https://cran.r-project.org/web/packages/fcfdr/index.html ) and demonstrate its utility in an application to type 1 diabetes, where we identify additional genetic associations. CONCLUSIONS: Our all-encompassing R package, fcfdr, serves as a comprehensive toolkit to unite GWAS and functional genomic data in order to increase statistical power to detect genetic associations.

Leveraging auxiliary data from arbitrary distributions to boost GWAS discovery with Flexible cFDR.

Genome-wide association studies (GWAS) have identified thousands of genetic variants that are associated with complex traits. However, a stringent significance threshold is required to identify robust genetic associations. Leveraging relevant auxiliary covariates has the potential to boost statistical power to exceed the significance threshold. Particularly, abundant pleiotropy and the non-random distribution of SNPs across various functional categories suggests that leveraging GWAS test statistics from related traits and/or functional genomic data may boost GWAS discovery. While type 1 error rate control has become standard in GWAS, control of the false discovery rate can be a more powerful approach. The conditional false discovery rate (cFDR) extends the standard FDR framework by conditioning on auxiliary data to call significant associations, but current implementations are restricted to auxiliary data satisfying specific parametric distributions, typically GWAS p-values for related traits. We relax these distributional assumptions, enabling an extension of the cFDR framework that supports auxiliary covariates from arbitrary continuous distributions ("Flexible cFDR"). Our method can be applied iteratively, thereby supporting multi-dimensional covariate data. Through simulations we show that Flexible cFDR increases sensitivity whilst controlling FDR after one or several iterations. We further demonstrate its practical potential through application to an asthma GWAS, leveraging various functional genomic data to find additional genetic associations for asthma, which we validate in the larger, independent, UK Biobank data resource.

Fine-mapping genetic associations.

Whilst thousands of genetic variants have been associated with human traits, identifying the subset of those variants that are causal requires a further 'fine-mapping' step. We review the basic fine-mapping approach, which is computationally fast and requires only summary data, but depends on an assumption of a single causal variant per associated region which is recognized as biologically unrealistic. We discuss different ways that the approach has been built upon to accommodate multiple causal variants in a region and to incorporate additional layers of functional annotation data. We further review methods for simultaneous fine-mapping of multiple datasets, either exploiting different linkage disequilibrium (LD) structures across ancestries or borrowing information between distinct but related traits. Finally, we look to the future and the opportunities that will be offered by increasingly accurate maps of causal variants for a multitude of human traits.

Debate: The pressing need for research and services for gender desisters/detransitioners.

The number of people presenting at gender clinics is increasing worldwide. Many people undergo a gender transition with subsequent improved psychological well-being (Paediatrics, 2014, 134, 696). However, some people choose to stop this journey, 'desisters', or to reverse their transition, 'detransitioners'. It has been suggested that some professionals and activists are reluctant to acknowledge the existence of desisters and detransitioners, possibly fearing that they may delegitimize persisters' experiences (International Journal of Transgenderism, 2018, 19, 231). Certainly, despite their presence in all follow-up studies of young people who have experienced gender dysphoria (GD), little thought has been given to how we might support this cohort. Levine (Archives of Sexual Behaviour, 2017, 47, 1295) reports that the 8th edition of the WPATH Standards of Care will include a section on detransitioning - confirming that this is an increasingly witnessed phenomenon worldwide. It also highlights that compared to the extensive protocols for working with children, adolescents and adults who wish to transition, nothing exists for those working with desisters or detransitioners. With very little research and no clear guidance on how to work with this population, and with numbers of referrals to gender services increasing, this is a timely juncture to consider factors that should be taken into account within clinical settings and areas for future research.

Resolving mechanisms of immune-mediated disease in primary CD4 T cells.

Deriving mechanisms of immune-mediated disease from GWAS data remains a formidable challenge, with attempts to identify causal variants being frequently hampered by strong linkage disequilibrium. To determine whether causal variants could be identified from their functional effects, we adapted a massively parallel reporter assay for use in primary CD4 T cells, the cell type whose regulatory DNA is most enriched for immune-mediated disease SNPs. This enabled the effects of candidate SNPs to be examined in a relevant cellular context and generated testable hypotheses into disease mechanisms. To illustrate the power of this approach, we investigated a locus that has been linked to six immune-mediated diseases but cannot be fine-mapped. By studying the lead expression-modulating SNP, we uncovered an NF-κB-driven regulatory circuit which constrains T-cell activation through the dynamic formation of a super-enhancer that upregulates TNFAIP3 (A20), a key NF-κB inhibitor. In activated T cells, this feedback circuit is disrupted-and super-enhancer formation prevented-by the risk variant at the lead SNP, leading to unrestrained T-cell activation via a molecular mechanism that appears to broadly predispose to human autoimmunity.

Improving the coverage of credible sets in Bayesian genetic fine-mapping.

Genome Wide Association Studies (GWAS) have successfully identified thousands of loci associated with human diseases. Bayesian genetic fine-mapping studies aim to identify the specific causal variants within GWAS loci responsible for each association, reporting credible sets of plausible causal variants, which are interpreted as containing the causal variant with some "coverage probability". Here, we use simulations to demonstrate that the coverage probabilities are over-conservative in most fine-mapping situations. We show that this is because fine-mapping data sets are not randomly selected from amongst all causal variants, but from amongst causal variants with larger effect sizes. We present a method to re-estimate the coverage of credible sets using rapid simulations based on the observed, or estimated, SNP correlation structure, we call this the "adjusted coverage estimate". This is extended to find "adjusted credible sets", which are the smallest set of variants such that their adjusted coverage estimate meets the target coverage. We use our method to improve the resolution of a fine-mapping study of type 1 diabetes. We found that in 27 out of 39 associated genomic regions our method could reduce the number of potentially causal variants to consider for follow-up, and found that none of the 95% or 99% credible sets required the inclusion of more variants-a pattern matched in simulations of well powered GWAS. Crucially, our method requires only GWAS summary statistics and remains accurate when SNP correlations are estimated from a large reference panel. Using our method to improve the resolution of fine-mapping studies will enable more efficient expenditure of resources in the follow-up process of annotating the variants in the credible set to determine the implicated genes and pathways in human diseases.

Recurrent HLA-B56 mediated neonatal alloimmune thrombocytopenia with fatal outcomes.

Neonatal alloimmune thrombocytopenia is caused by maternal antibodies against paternally-inherited alloantigens present on foetal platelets and is a significant cause of morbidity and mortality in neonates. While generally thought of as a human platelet antibody mediated phenomenon, cases of HLA mediated NAIT have been reported. We document the investigation of a patient with two pregnancies affected by HLA-B56 mediated NAIT and a proposal for management of future pregnancies.

A Case of an Acquired Factor VIII Inhibitor Complicated by Multiple Treatment-Related Opportunistic Infections and Review of the Literature.

This case report describes a patient with an idiopathic acquired Factor VIII inhibitor and severe bleeding. She was treated with rituximab after failing first-line treatment with steroids and cyclophosphamide. Two months following rituximab treatment, our patient developed a succession of severe opportunistic infections requiring intensive care unit admission. Over a period of 12 weeks she required treatment for Pseudomonas aeruginosa septicaemia, herpes simplex gingivostomatitis and pharyngotonsillitis, clostridium difficile-related diarrhoea, systemic cytomegalovirus infection, pneumocystis jiroveci, and invasive pulmonary aspergillosis lung infections. After significant rehabilitation, the patient was finally discharged following a 5-month admission. This case highlights the complexity of balancing a life-threatening condition with the side effects of treatment. It also raises the issue of routine prophylaxis for immunosuppression in nonmalignant conditions, which will become a common dilemma with the expanding indications for rituximab use.

Neurobiological status at initial presentation predicts neuropsychological functioning in early onset anorexia nervosa at four-year follow up.

This study explores whether neurobiological status (indexed by regional cerebral blood flow) at initial presentation predicts neuropsychological status at four-year follow up in a sample of children with early onset anorexia nervosa. Neuropsychological assessment was conducted on 15 females four years after their initial treatment, and matched controls. At follow up there were significant differences between subgroups (based on neurobiological status at initial presentation) and matched controls in long-term visual memory and cognitive inhibition. This study offers preliminary evidence that neurobiological abnormalities at initial presentation predict neuropsychological status at follow up, suggesting a distinct neurodevelopmental subtype of early onset anorexia nervosa.

How should paediatricians assess Gillick competence?

Competence is an essential legal requirement for valid consent to medical treatment. Children under 16 may be considered 'Gillick competent' to make treatment decisions, but may need to demonstrate this. Applied tests for competence are wide-ranging and context dependent. Competence is related to cognitive ability and experience and may be enhanced by education, encouragement etc.; there is a general duty for professionals to enhance the competence of children in their care. The need to assess a child's competence may occur when s/he wishes to make a controversial decision whose wisdom others dispute. Potential assessors should have the necessary practical skills and an understanding of the child in their social and medical context. Assessments should be developmentally appropriate, explore systemic influences, and consider the child's emotional state, cognitive development and ability to balance risks and benefits. The involvement of a psychologist or other independent third party should be considered in cases that raise serious concerns about competency, or that involve complex decisions or conflict between the various parties. In rare cases courts may be involved.

Spatial variation of haemosporidian parasite infection in African rainforest bird species.

Spatial heterogeneity influences the distribution, prevalence, and diversity of haemosporidian parasites. Previous studies have found complex patterns of prevalence with respect to habitat characteristics and parasite genotype, and their interactions, but there is little information regarding how parasitemia intensity and the prevalence of co-infections may vary in space. Here, using both molecular methods and microscopy, we report an analysis of the variation of parasitemia intensity and co-infections of avian haemosporidian parasites ( Plasmodium and Haemoproteus species) in 2 common African birds species, the yellow-whiskered greenbul ( Andropadus latirostris ) and the olive sunbird ( Cyanomitra olivacea ), at 3 sites with distinct habitat characteristics in Ghana. First, we found an interaction between the site and host species for the prevalence of Plasmodium spp. and Haemoproteus spp. For the olive sunbird, the prevalence of Plasmodium spp., as well as the number of individuals with co-infections, varied significantly among the sites, but these measures remained constant for the yellow-whiskered greenbul. In addition, yellow-whiskered greenbuls infected with Haemoproteus spp. were found only at 1 site. Furthermore, for both bird species, the parasitemia intensity of Plasmodium spp. varied significantly among the 3 sites, but with opposing trends. These results suggest that spatial heterogeneity differently affects haemosporidian infection parameters in these vertebrate-hosts. Environmental conditions here can either favor or reduce parasite infection. We discuss the implications of these discrepancies for conservation and ecological studies of infectious diseases in natural populations.