Evolution-based strategies to elucidate genotype-phenotype relationships in traits relevant to human health

Understanding the link between genotype and phenotype is a key question in biological research. In other words, how do changes in DNA sequence give rise to the enormous diversity of life on Earth? In this work, we address that question by focusing on the genomics of convergent phenotypes, or phenotypes that have evolved independently in unrelated species. These natural biological replicates of phenotype evolution allow us to identify genomic regions, either regulatory or protein coding, that have experienced convergent evolution in concordance with convergent evolution of phenotypes, thus linking genomic regions to phenotypes. In this work, we calculate evolutionary rates throughout the mammalian phylogeny for numerous genomic sequences to find concordance between species phenotype and evolutionary rate of sequences to link genomic regions to phenotypes. Chapter one describes three methods associated with quantifying the connection between genomic region evolution and phenotype evolution. First, RERconverge connects genomic regions to phenotypes in a linear regression-based framework. Second, permulations are a statistical extension to RERconverge that allow for rigorous calculation of confidence in associations from RERconverge. Third, proper implementation of branch-site models for convergent positive selection allows for identification of genes potentially driving convergent evolution. Chapter two describes implementation of methods from chapter one to longevity phenotypes in 61 mammal species. We found increased evolutionary constraint in cancer control genes in large, long-lived species, thus likely conferring additional protection from cancer. Species exceptionally long-lived given their size showed increased evolutionary constraint on DNA repair pathways, indicating that efficient DNA repair is important to evolution of extreme lifespan independent of body size. This work provided insight into pan-mammalian genomic mechanisms underlying lifespan. Chapter three describes further implementation of chapter one methods to the hairlessness phenotype in 61 mammal species. Although all mammals have some hair at some developmental time point, several mammals, such as cetaceans, naked mole-rats, armadillos, and humans, have relatively little hair. Many genomic elements we identified were known to be hair-related, and many more are valuable candidates for further testing into hair-related functions. This work for the first time provided insights to the natural evolution of mammalian hairlessness

User experience analysis of AbC-19 Rapid Test via lateral flow immunoassays for self-administrated SARS-CoV-2 antibody testing

Abstract Lateral flow immunoassays are low cost, rapid and highly efficacious point-of-care devices, which have been used for SARS-CoV-2 antibody testing by professionals. However, there is a lack of understanding about how self-administered tests are used by the general public for mass testing in different environmental settings. The purpose of this study was to assess the user experience (UX) (including usability) of a self-testing kit to identify COVID-19 antibodies used by a representative sample of the public in their cars, which included 1544 participants in Northern Ireland. The results based on 5-point Likert ratings from a post-test questionnaire achieved an average UX score of 96.03% [95% confidence interval (CI) 95.05–97.01%], suggesting a good degree of user experience. The results of the Wilcoxon rank sum tests suggest that UX scores were independent of the user’s age and education level although the confidence in this conclusion could be strengthened by including more participants aged younger than 18 and those with only primary or secondary education. The agreement between the test result as interpreted by the participant and the researcher was 95.85% [95% CI 94.85–96.85%], Kappa score 0.75 [95% CI 0.69–0.81] (indicating substantial agreement). Text analysis via the latent Dirichlet allocation model for the free text responses in the survey suggest that the user experience could be improved for blood-sample collection, by modifying the method of sample transfer to the test device and giving clearer instructions on how to interpret the test results. The overall findings provide an insight into the opportunities for improving the design of SARS-CoV-2 antibody testing kits to be used by the general public and therefore inform protocols for future user experience studies of point-of-care tests

Mutation-independent Allele-Specific Editing by CRISPR-Cas9, a Novel Approach to Treat Autosomal Dominant Disease

CRISPR-Cas9 provides a tool to treat autosomal dominant disease by non-homologous end joining (NHEJ) gene disruption of the mutant allele. In order to discriminate between wild-type and mutant alleles, Streptococcus pyogenes Cas9 (SpCas9) must be able to detect a single nucleotide change. Allele-specific editing can be achieved by using either a guide-specific approach, in which the missense mutation is found within the guide sequence, or a protospacer-adjacent motif (PAM)-specific approach, in which the missense mutation generates a novel PAM. While both approaches have been shown to offer allele specificity in certain contexts, in cases where numerous missense mutations are associated with a particular disease, such as TGFBI (transforming growth factor β-induced) corneal dystrophies, it is neither possible nor realistic to target each mutation individually. In this study, we demonstrate allele-specific CRISPR gene editing independent of the disease-causing mutation that is capable of achieving complete allele discrimination, and we propose it as a targeting approach for autosomal dominant disease. Our approach utilizes natural variants in the target region that contain a PAM on one allele that lies in cis with the causative mutation, removing the constraints of a mutation-dependent approach. Our innovative patient-specific guide design approach takes into account the patient’s individual genetic make-up, allowing on- and off-target activity to be assessed in a personalized manner

Ancient convergent losses of Paraoxonase 1 yield potential risks for modern marine mammals

Mammals diversified by colonizing drastically different environments, with each transition yielding numerous molecular changes, including losses of protein function. Though not initially deleterious, these losses could subsequently carry deleterious pleiotropic consequences. We have used phylogenetic methods to identify convergent functional losses across independent marine mammal lineages. In one extreme case, Paraoxonase 1 (PON1) accrued lesions in all marine lineages, while remaining intact in all terrestrial mammals. These lesions coincide with PON1 enzymatic activity loss in marine species’ blood plasma. This convergent loss is likely explained by parallel shifts in marine ancestors’ lipid metabolism and/or bloodstream oxidative environment affecting PON1’s role in fatty acid oxidation. PON1 loss also eliminates marine mammals’ main defense against neurotoxicity from specific man-made organophosphorus compounds, implying potential risks in modern environment

Ancient convergent losses of Paraoxonase 1 yield potential risks for modern marine mammals

Mammals diversified by colonizing drastically different environments, with each transition yielding numerous molecular changes, including losses of protein function. Though not initially deleterious, these losses could subsequently carry deleterious pleiotropic consequences. We have used phylogenetic methods to identify convergent functional losses across independent marine mammal lineages. In one extreme case, Paraoxonase 1 (PON1) accrued lesions in all marine lineages, while remaining intact in all terrestrial mammals. These lesions coincide with PON1 enzymatic activity loss in marine species’ blood plasma. This convergent loss is likely explained by parallel shifts in marine ancestors’ lipid metabolism and/or bloodstream oxidative environment affecting PON1’s role in fatty acid oxidation. PON1 loss also eliminates marine mammals’ main defense against neurotoxicity from specific man-made organophosphorus compounds, implying potential risks in modern environment

Evaluation of the IgG antibody response to SARS CoV-2 infection and performance of a lateral flow immunoassay: cross-sectional and longitudinal analysis over 11 months

OBJECTIVE: To evaluate the dynamics and longevity of the humoral immune response to SARS-CoV-2 infection and assess the performance of professional use of the UK-RTC AbC-19 Rapid Test lateral flow immunoassay (LFIA) for the target condition of SARS-CoV-2 spike protein IgG antibodies. DESIGN: Nationwide serological study. SETTING: Northern Ireland, UK, May 2020–February 2021. PARTICIPANTS: Plasma samples were collected from a diverse cohort of individuals from the general public (n=279), Northern Ireland healthcare workers (n=195), pre-pandemic blood donations and research studies (n=223) and through a convalescent plasma programme (n=183). Plasma donors (n=101) were followed with sequential samples over 11 months post-symptom onset. MAIN OUTCOME MEASURES: SARS-CoV-2 antibody levels in plasma samples using Roche Elecsys Anti-SARS-CoV-2 IgG/IgA/IgM, Abbott SARS-CoV-2 IgG and EuroImmun IgG SARS-CoV-2 ELISA immunoassays over time. UK-RTC AbC-19 LFIA sensitivity and specificity, estimated using a three-reference standard system to establish a characterised panel of 330 positive and 488 negative SARS-CoV-2 IgG samples. RESULTS: We detected persistence of SARS-CoV-2 IgG antibodies for up to 10 months post-infection, across a minimum of two laboratory immunoassays. On the known positive cohort, the UK-RTC AbC-19 LFIA showed a sensitivity of 97.58% (95.28% to 98.95%) and on known negatives, showed specificity of 99.59% (98.53 % to 99.95%). CONCLUSIONS: Through comprehensive analysis of a cohort of pre-pandemic and pandemic individuals, we show detectable levels of IgG antibodies, lasting over 46 weeks when assessed by EuroImmun ELISA, providing insight to antibody levels at later time points post-infection. We show good laboratory validation performance metrics for the AbC-19 rapid test for SARS-CoV-2 spike protein IgG antibody detection in a laboratory-based setting

Dissociable effects of cannabis with and without cannabidiol on the human brain’s resting-state functional connectivity

Background: Two major constituents of cannabis are Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). THC is the main psychoactive component; CBD may buffer the user against the harmful effects of THC. Aims: We examined the effects of two strains of cannabis and placebo on the human brain’s resting-state networks using fMRI. Methods: Seventeen healthy volunteers (experienced with cannabis, but not regular users) underwent three drug treatments and scanning sessions. Treatments were cannabis containing THC (Cann−CBD; 8 mg THC), cannabis containing THC with CBD (Cann+CBD; 8 mg THC + 10 mg CBD), and matched placebo cannabis. Seed-based resting-state functional connectivity analyses were performed on three brain networks: the default mode (DMN; defined by positive connectivity with the posterior cingulate cortex: PCC+), executive control (ECN; defined by negative connectivity with the posterior cingulate cortex: PCC−) and salience (SAL; defined by positive connectivity with the anterior insula: AI+) network. Results: Reductions in functional connectivity (relative to placebo) were seen in the DMN (PCC+) and SAL (AI+) networks for both strains of cannabis, with spatially dissociable effects. Across the entire salience network (AI+), Cann−CBD reduced connectivity relative to Cann+CBD. The PCC in the DMN was specifically disrupted by Cann−CBD, and this effect correlated with subjective drug effects, including feeling ‘stoned’ and ‘high’. Conclusions: THC disrupts the DMN, and the PCC is a key brain region involved in the subjective experience of THC intoxication. CBD restores disruption of the salience network by THC, which may explain its potential to treat disorders of salience such as psychosis and addiction

Vocal learning-associated convergent evolution in mammalian proteins and regulatory elements.

Vocal production learning (vocal learning) is a convergently evolved trait in vertebrates. To identify brain genomic elements associated with mammalian vocal learning, we integrated genomic, anatomical, and neurophysiological data from the Egyptian fruit bat (Rousettus aegyptiacus) with analyses of the genomes of 215 placental mammals. First, we identified a set of proteins evolving more slowly in vocal learners. Then, we discovered a vocal motor cortical region in the Egyptian fruit bat, an emergent vocal learner, and leveraged that knowledge to identify active cis-regulatory elements in the motor cortex of vocal learners. Machine learning methods applied to motor cortex open chromatin revealed 50 enhancers robustly associated with vocal learning whose activity tended to be lower in vocal learners. Our research implicates convergent losses of motor cortex regulatory elements in mammalian vocal learning evolution