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Glycophorins are transmembrane proteins of red blood cells (RBCs), heavily glycosylated on their external-facing surface. In humans, there are four glycophorin proteins, glycophorins A, B, C and D. Glycophorins A and B are encoded by two similar genes GYPA and GYPB, and glycophorin C and glycophorin D are encoded by a single gene, GYPC. The exact function of glycophorins remains unclear. However, given their abundance on the surface of RBCs, it is likely that they serve as a substrate for glycosylation, giving the RBC a negatively charged, complex glycan “coat”. GYPB and GYPE (a closely related pseudogene) were generated from GYPA by two duplication events involving a 120-kb genomic segment between 10 and 15 million years ago. Non-allelic homologous recombination between these 120-kb repeats generates a variety of duplication alleles and deletion alleles, which have been systematically catalogued from genomic sequence data. One allele, called DUP4, encodes the Dantu NE blood type and is strongly protective against malaria as it alters the surface tension of the RBC membrane. Glycophorins interact with other infectious pathogens, including viruses, as well as the malarial parasite Plasmodium falciparum, but the role of glycophorin variation in mediating the effects of these pathogens remains underexplored.

Malignant pleural mesothelioma (MPM), a rare cancer a long latency period (up to 40 years) between asbestos exposure and disease presentation. The mechanisms coupling asbestos to recurrent somatic alterations are poorly defined. Gene-fusions arising through genomic instability may create novel drivers during early MPM evolution. We explored the gene fusions that occurred early in the evolutionary history. We conducted multiregional whole exome sequencing (WES) of 106 samples from 20 patients undergoing pleurectomy decortication and identified 24 clonal non-recurrent fusions, three of which were novel (FMO9P-OR2W5,GBA3andSP9). The number of early gene fusion events detected varied from zero to eight per tumour, and presence of gene fusions was associated with clonal SCNAs involving the Hippo pathway genes and homologous recombination DNA repair genes. Fusions involved known tumour suppressorsBAP1,MTAP, andLRP1B,and a clonal oncogenic fusion involvingCACNA1D-ERC2, PARD3B-NT5DC2andSTAB2-NT5DC2fusions were also identified as clonal fusions. Gene fusions events occur early during MPM evolution. Individual fusions are rare as no recurrent truncal fusions event were found. This suggests the importance of early disruption of these pathways in generating genomic rearrangements resulting in potentially oncogenic gene fusions.

Summary DeepPheWAS is an R package for phenome-wide association studies that creates clinically curated composite phenotypes and integrates quantitative phenotypes from primary care data, longitudinal trajectories of quantitative measures, disease progression and drug response phenotypes. Tools are provided for efficient analysis of association with any genetic input, under any genetic model, with optional sex-stratified analysis, and for developing novel phenotypes. Availability and implementation The DeepPheWAS R package is freely available under GNU general public licence v3.0 from at https://github.com/Richard-Packer/DeepPheWAS. Supplementary information Supplementary data are available at Bioinformatics online.

Structural variation (SV) is a large difference (typically >100 bp) in the genomic structure of two genomes and includes both copy number variation and variation that does not change copy number of a genomic region, such as an inversion. Improved reference genomes, combined with widespread genome sequencing using short-read sequencing technology, and increasingly using long-read sequencing, have reignited interest in SV. Recent large-scale studies and functional focused analyses have highlighted the role of SV in human evolution. In this review, we highlight human-specific SVs involved in changes in the brain, population-specific SVs that affect response to the environment, including adaptation to diet and infectious diseases, and summarise the contribution of archaic hominin admixture to present-day human SV.

Intelectins are a family of multimeric secreted proteins that bind microbe-specific glycans. Both genetic and functional studies have suggested that intelectins have an important role in innate immunity and are involved in the etiology of various human diseases, including inflammatory bowel disease. Experiments investigating the role of intelectins in human disease using mouse models are limited by the fact that there is not a clear one-to-one relationship between intelectin genes in humans and mice, and that the number of intelectin genes varies between different mouse strains. In this study we show by gene sequence and gene expression analysis that human intelectin-1 (ITLN1) has multiple orthologues in mice, including a functional homologue Itln1; however, human intelectin-2 has no such orthologue or homologue. We confirm that all sub-strains of the C57 mouse strain have a large deletion resulting in retention of only one intelectin gene, Itln1. The majority of laboratory strains have a full complement of six intelectin genes, except CAST, SPRET, SKIVE, MOLF and PANCEVO strains, which are derived from different mouse species/subspecies and encode different complements of intelectin genes. In wild mice, intelectin deletions are polymorphic in Mus musculus castaneus and Mus musculus domesticus. Further sequence analysis shows that Itln3 and Itln5 are polymorphic pseudogenes due to premature truncating mutations, and that mouse Itln1 has undergone recent adaptive evolution. Taken together, our study shows extensive diversity in intelectin genes in both laboratory and wild-mice, suggesting a pattern of birth-and-death evolution. In addition, our data provide a foundation for further experimental investigation of the role of intelectins in disease.

SummaryACE inhibitors (ACEIs) are commonly prescribed for hypertension, a global risk factor for cardiovascular disease. Their primary side effect is a dry cough which affects 5-35% of users. As clinical guidelines recommend switching those experiencing cough to an angiotensin-II receptor blocker, we have used this switch as a proxy for ACEI-induced cough. Through a two-stage multi-ancestry genome-wide association study, including up to 7,030 cases and 39,921 controls, we identify five independent genome-wide significant associations implicating six protein-coding genes, including INHBC, KCNIP4, NTSR1 and PREP which encode proteins involved in the nervous system. We also observe genetic overlap between ACEI-induced cough and chronic dry cough through genetic correlation and phenome-wide association studies. In line with existing hypotheses, our findings suggest a neurological basis for the pathology of ACEI-induced cough, particularly the role of proinflammatory mediators in sensory airway sensitivity and cough reflex modulation, and shared biological mechanisms with chronic dry cough.

Short tandem repeat (STR) variation is an often overlooked source of variation between genomes. STRs comprise about 3% of the human genome sequence, are highly polymorphic, and are known to cause not only Mendelian disease, but extensively affect gene expression. Nevertheless, their contribution to common disease is not well-understood, but recent software tools designed to genotype STRs using short read sequencing data are beginning to address this. Here, we compare software that genotypes common STRs and detect rarer STR expansions genome-wide, with the aim of applying them to population-scale genome sequences. By using the Genome-In-A-Bottle (GIAB) consortium and 1000 Genomes Project sequencing data, we can compare performance in terms of sequence length, depth, computing resources needed, genotyping accuracy and number of STRs genotyped. To ensure broad applicability of our findings, we also measure performance against a set of clinical samples with known STR expansions, and a set of STRs commonly used for forensic identification. By using analysis of Mendelian inheritance patterns and comparison with capillary electrophoresis genotypes, we find that both HipSTR and GangSTR perform well in genotyping common STRs, with GangSTR outperforming HipSTR for genotyping call rate and memory usage. Analysis for expanded STRs showed ExpansionHunter denovo (EHdn) and STRetch outperformed GangSTR, but EHdn used considerably less processor time and memory compared to STRetch. Analysis on shared genomic sequence data provided by the GIAB consortium allows future performance comparisons of new software approaches on a common set of data, facilitating comparisons and allowing researchers to choose the best software that fulfils their needs.

Intelectins (intestinal lectins) are highly conserved across chordate evolution and have been implicated in various human diseases, including Crohn's disease (CD). The human genome encodes two intelectin genes, intelectin-1 (ITLN1) and intelectin-2 (ITLN2). Other than its high sequence similarity with ITLN1, little is known about ITLN2. To address this void in knowledge, we report that ITLN2 exhibits discrete, yet notable differences from ITLN1 in primary structure, including a unique amino terminus, as well as changes in amino acid residues associated with the glycan-binding activity of ITLN1. We identified that ITLN2 is a highly abundant Paneth cell-specific product, which localizes to secretory granules, and is expressed as a multimeric protein in the small intestine. In surgical specimens of ileal CD, ITLN2mRNA levels were reduced approximately five-fold compared to control specimens. The ileal expression of ITLN2 was unaffected by previously reported disease-associated variants in ITLN2 and CD-associated variants in neighboring ITLN1 as well as NOD2 and ATG16L1. ITLN2mRNA expression was undetectable in control colon tissue; however, in both ulcerative colitis (UC) and colonic CD, metaplastic Paneth cells were found to express ITLN2. Together, the data reported establish the groundwork for understanding ITLN2 function(s) in the intestine, including its possible role in CD.

Structural variation in the human genome can affect risk of disease. An example is a complex structural variant of the human glycophorin gene cluster, called DUP4, which is associated with a clinically significant level of protection against severe malaria. The human glycophorin gene cluster harbours at least 23 distinct structural variants, and accurate genotyping of this complex structural variation remains a challenge. Here, we use a polymerase chain reaction-based strategy to genotype structural variation at the human glycophorin gene cluster, including the alleles responsible for the U- blood group. We validate our approach, based on a triplex paralogue ratio test, on publically available samples from the 1000 Genomes project. We then genotype 574 individuals from a longitudinal birth cohort (Tori-Bossito cohort) using small amounts of DNA at low cost. Our approach readily identifies known deletions and duplications, and can potentially identify novel variants for further analysis. It will allow exploration of genetic variation at the glycophorin locus, and investigation of its relationship with malaria, in large sample sets at minimal cost, using standard molecular biology equipment.

Discovering loci under balancing selection in humans can identify loci with alleles that affect response to the environment and disease. Genome variation data have identified the 5' region of the

Most genetic variation in humans occurs in a pattern consistent with neutral evolution, but a small subset is maintained by balancing selection. Identifying loci under balancing selection is important not only for understanding the processes explaining variation in the genome, but also to identify loci with alleles that affect response to the environment and disease. Several genome scans using genetic variation data have identified the 5’ end of the DMBT1 gene as a region undergoing balancing selection. DMBT1 encodes the pattern-recognition glycoprotein DMBT1, also known as SALSA, gp340 or salivary agglutinin. It binds to a wide variety of pathogens through a tandemly-arranged scavenger receptor cysteine-rich (SRCR) domain, with the number of SRCR domains varying in humans. Here we use expression analysis, linkage in pedigrees, and long range single transcript sequencing, to show that the signal of balancing selection is driven by one haplotype usually carrying shorter SRCR repeats, and another usually carrying a longer SRCR repeat, within the coding region of DMBT1. The DMBT1 protein size isoform encoded by a shorter SRCR domain repeat allele showed complete loss of binding of a cariogenic and invasive Streptococcus mutans strain in contrast to the long SRCR allele. Taken together, our results suggest that balancing selection at DMBT1 is due to host-microbe interactions of encoded SRCR tandem repeat alleles.

Background and aimsNon-alcoholic fatty liver disease (NAFLD) is a complex trait that has a global prevalence estimated as 25%. We aimed to identify the genetic variant underlying a four-generation family with progressive NAFLD leading to cirrhosis, decompensation and development of hepatocellular carcinoma in the absence of common risk factors such as obesity and type 2 diabetes.MethodsExome sequencing and genome comparisons were used to identify the likely causal variant. We extensively characterised the clinical phenotype and post-prandial metabolic responses of family members with the identified novel variant in comparison to healthy non-carriers and wild type patients with NAFLD. Variant-expressing hepatocyte-like cells (HLCs) were derived from human induced pluripotent stem cells generated from homozygous donor skin fibroblasts. The phenotype was assessed using imaging, targeted RNA analysis and molecular expression arrays.ResultsWe identified a rare causal variant in MTTP, c.1691T>C p.I564T (rs745447480) encoding microsomal triglyceride transfer protein (MTP) associated with progressive non-alcoholic fatty liver disease, unrelated to metabolic syndrome. Although other described mutations in MTTP cause abetalipoproteinemia, neither homozygotes nor heterozygotes exhibited characteristic manifestations of this severe disease. HLCs derived from a homozygote donor had lower lipoprotein ApoB secretion, compared to wild type cells. Cytoplasmic triglyceride accumulation in HLCs triggered endoplasmic reticulum stress, secretion of pro-inflammatory mediators and production of reactive oxygen species.ConclusionWe have identified and characterized a rare causal variant in MTTP and homozygosity for MTTP p.I564T is associated with progressive NAFLD without any other manifestations of abetalipoproteinemia.

Intelectins are ancient carbohydrate binding proteins, spanning chordate evolution and implicated in multiple human diseases. Previous GWAS have linked SNPs in ITLN1 (also known as omentin) with susceptibility to Crohn's disease (CD); however, analysis of possible functional significance of SNPs at this locus is lacking. Using the Ensembl database, pairwise linkage disequilibrium (LD) analyses indicated that several disease-associated SNPs at the ITLN1 locus, including SNPs in CD244 and Ly9, were in LD. The alleles comprising the risk haplotype are the major alleles in European (67%), but minor alleles in African superpopulations. Neither ITLN1 mRNA nor protein abundance in intestinal tissue, which we confirm as goblet-cell derived, was altered in the CD samples overall nor when samples were analyzed according to genotype. Moreover, the missense variant V109D does not influence ITLN1 glycan binding to the glycan β-D-galactofuranose or protein–protein oligomerization. Taken together, our data are an important step in defining the role(s) of the CD-risk haplotype by determining that risk is unlikely to be due to changes in ITLN1 carbohydrate recognition, protein oligomerization, or expression levels in intestinal mucosa. Our findings suggest that the relationship between the genomic data and disease arises from changes in CD244 or Ly9 biology, differences in ITLN1 expression in other tissues, or an alteration in ITLN1 interaction with other proteins.

MPM is a pleural tumour associated with asbestos exposure. There is a long latency period of more than 40 years between asbestos exposure and disease presentation, highlighting an unknown evolutionary trajectory. Throughout the latent years, different somatic structural alterations are acquired by the growing tumour. To elucidate this evolutionary path, we conducted multiregional whole exome sequencing (WES) of 106 samples (5 or 4 regions per tumour and a matched blood sample) for 20 patients as part of the MEDUSA study (Mesothelioma evolution: Drugging somatic alterations). Gene fusions have been shown to have a clinical, diagnostic, and therapeutic significance i.e., the driver fusion EM4-ALK expressed in a minority of peritoneal mesotheliomas. Previous descriptions of gene fusions in MPM have mostly been limited to isolated case reports, with the only genome-wide assessment study identifying fusion events involving known MPM genes including NF2 and BAP1. Therefore, our aim was to identify potential truncal inter- and intra-chromosomal rearrangements generating gene fusions that occurred early in the evolutionary history of the tumour. We used two software tools - Meerkat (v.0.189) and Delly (v0.8.7), to identify gene fusion events in the regional tumour sites for a given patient. We identified tumour-specific events by filtering against the calls made on exome sequence from the matched blood of each patient. Finally, we identified truncal gene fusion events as ones observed across all regional samples of a particular patient. We validated the gene fusions by PCR and Sanger sequencing of the breakpoint junctions. Further work was carried out to identify truncal fusion transcripts at the transcriptomic level using RNA sequencing data (Arriba v2.1.0) and compare the gene fusion predictions detected at the genomic level made by WES. We found a total of 24 truncal fusion events, 3 of which were novel (FMO9P-OR2W5, GBA3 and SP9). Tumour suppressor genes involved included (BAP1, MTAP, and LRP1B). Oncogenic fusions involved (CACNA1D-ERC2). In addition to this, our analysis revealed truncal copy number neutral events such as in-frame PARD3B-NT5DC2 and out of frame STAB2-NT5DC2. Moreover, with transcriptome sequencing we characterised the fusion transcripts events initially detected by WES (i.e. fusions involving AHRR and B3GAL2 genes). Our findings suggest that gene fusions events occur during early evolution with marked heterogeneity across the cohort (no recurrent truncal fusions event were observed in a subset). Some truncal fusion events are likely to be drivers of cancer progression, as they involve mesothelioma associated genes (e.g. MTAP and BAP1). The role of other gene fusions remains unclear, and larger studies are needed to clarify whether somatic rearrangements of these genes are involved in tumour progression in MPM. Citation Format: Maymun Jama, Edward J Hollox, Apostolos Nakas, Lee Brannan, Alan Dawson, frank dudbridge, Dean Fennell. Mesotheliomas harbor early clonal fusions involving both tumor suppressor drivers and novel oncogenic alterations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5693.

Variability in SARS-CoV-2 susceptibility and COVID-19 disease severity between individuals is partly due to genetic factors. Here, we identify 4 genomic loci with suggestive associations for SARS-CoV-2 susceptibility and 19 for COVID-19 disease severity. Four of these 23 loci likely have an ethnicity-specific component. Genome-wide association study (GWAS) signals in 11 loci colocalize with expression quantitative trait loci (eQTLs) associated with the expression of 20 genes in 62 tissues/cell types (range: 1:43 tissues/gene), including lung, brain, heart, muscle, and skin as well as the digestive system and immune system. We perform genetic fine mapping to compute 99% credible SNP sets, which identify 10 GWAS loci that have eight or fewer SNPs in the credible set, including three loci with one single likely causal SNP. Our study suggests that the diverse symptoms and disease severity of COVID-19 observed between individuals is associated with variants across the genome, affecting gene expression levels in a wide variety of tissue types., Graphical abstract, D’Antonio et al. characterize associations between GWAS signals for COVID-19 disease and eQTLs in 69 human tissues to identify causal variants and their underlying molecular mechanisms. They show that diverse symptoms and disease severity of COVID-19 are associated with variants affecting gene expression in a wide variety of tissues.

A persistent unexplained eosinophilia is relatively unusual and has a number of causes of which allergy and infection with helminthic parasites are the most common diagnoses. Less common causes include idiopathic hypereosinophilic syndrome (HES), eosinophilic granulomatosis with polyangiitis (EGPA) and single organ conditions such as eosinophilic gastrointestinal disease (EGID) (1) . Although occasionally caused by myeloproliferative neoplasm in most cases eosinophilia is due to excess production of growth factors, particularly interleukin-5, by bystander cells which are often lymphoid in nature. Sometimes an environmental trigger can be identified, but the underlying cause of the eosinophilia often remains obscure.

Intelectins are a family of multimeric secreted proteins that bind microbe-specific glycans. Both genetic and functional studies have suggested that intelectins have an important role in innate immunity and are involved in the etiology of various human diseases, including inflammatory bowel disease. Experiments investigating the role of intelectins in human disease using mouse models are limited by the fact that there is not a clear one-to-one relationship between intelectin genes in humans and mice, and that the number of intelectin genes varies between different mouse strains. In this study we show by gene sequence and gene expression analysis that human intelectin-1 (ITLN1) has multiple orthologues in mice, including a functional homologue Itln1; however, human intelectin-2 has no such orthologue or homologue. We confirm that all sub-strains of the C57-line have a large deletion resulting in retention of only one intelectin gene, Itln1. The majority of laboratory strains have a full complement of six intelectin genes, except wild-derived CAST, SPRET, SKIVE, MOLF and PANCEVO, which are derived from different mouse species/subspecies and encode different complements of intelectin genes. In wild mice, intelectin deletions are polymorphic in Mus musculus castaneus and Mus musculus domesticus. Further sequence analysis shows that Itln3 and Itln5 are polymorphic pseudogenes due to premature truncating mutations, and that mouse Itln1 has undergone recent adaptive evolution. Taken together, our study shows extensive diversity in intelectin genes in both laboratory and wild-mice, suggesting a pattern of birth-and-death evolution. In addition, our data provide a foundation for further experimental investigation of the role of intelectins in disease.

Malignant Pleural Mesothelioma (MPM) is typically diagnosed 20–50 years after exposure to asbestos and evolves along an unknown evolutionary trajectory. To elucidate this path, we conducted multi-regional exome sequencing of 90 tumour samples from 22 MPMs acquired at surgery. Here we show that exomic intratumour heterogeneity varies widely across the cohort. Phylogenetic tree topology ranges from linear to highly branched, reflecting a steep gradient of genomic instability. Using transfer learning, we detect repeated evolution, resolving 5 clusters that are prognostic, with temporally ordered clonal drivers. BAP1/−3p21 and FBXW7/-chr4 events are always early clonal. In contrast, NF2/−22q events, leading to Hippo pathway inactivation are predominantly late clonal, positively selected, and when subclonal, exhibit parallel evolution indicating an evolutionary constraint. Very late somatic alteration of NF2/22q occurred in one patient 12 years after surgery. Clonal architecture and evolutionary clusters dictate MPM inflammation and immune evasion. These results reveal potentially drugable evolutionary bottlenecking in MPM, and an impact of clonal architecture on shaping the immune landscape, with potential to dictate the clinical response to immune checkpoint inhibition., The impact of intratumour heterogeneity on immune surveillance and clinical outcomes has not been adequately explored in malignant pleural mesothelioma (MPM). Here the authors analyse the influence of evolution on the survival and immune landscape of MPM patients using multi-region sequencing data.

Background Approximately 5% of the human genome shows common structural variation, which is enriched for genes involved in the immune response and cell-cell interactions. A well-established region of extensive structural variation is the glycophorin gene cluster, comprising three tandemly-repeated regions about 120 kb in length and carrying the highly homologous genes GYPA, GYPB and GYPE. Glycophorin A (encoded by GYPA) and glycophorin B (encoded by GYPB) are glycoproteins present at high levels on the surface of erythrocytes, and they have been suggested to act as decoy receptors for viral pathogens. They are receptors for the invasion of the protist parasite Plasmodium falciparum, a causative agent of malaria. A particular complex structural variant, called DUP4, creates a GYPB-GYPA fusion gene known to confer resistance to malaria. Many other structural variants exist across the glycophorin gene cluster, and they remain poorly characterised. Results Here, we analyse sequences from 3234 diploid genomes from across the world for structural variation at the glycophorin locus, confirming 15 variants in the 1000 Genomes project cohort, discovering 9 new variants, and characterising a selection of these variants using fibre-FISH and breakpoint mapping at the sequence level. We identify variants predicted to create novel fusion genes and a common inversion duplication variant at appreciable frequencies in West Africans. We show that almost all variants can be explained by non-allelic homologous recombination and by comparing the structural variant breakpoints with recombination hotspot maps, confirm the importance of a particular meiotic recombination hotspot on structural variant formation in this region. Conclusions We identify and validate large structural variants in the human glycophorin A-B-E gene cluster which may be associated with different clinical aspects of malaria.

Background: New data collection in established longitudinal population studies provides an opportunity for studying the risk factors and sequelae of the novel coronavirus disease 2019 (COVID-19), plus the indirect impacts of the COVID-19 pandemic on wellbeing. The Extended Cohort for E-health, Environment and DNA (EXCEED) cohort is a population-based cohort (N>11,000), recruited from 2013 in Leicester, Leicestershire and Rutland. EXCEED includes consent for electronic healthcare record (EHR) linkage, spirometry, genomic data, and questionnaire data. Methods: Between May 2020 and July 2021, a new questionnaire was deployed in EXCEED, which captured COVID-19 symptoms, general physical and mental health, plus socioeconomic and environmental factors during the pandemic. An online system was developed to invite new participants to join EXCEED, with informed consent being provided online. New and existing participants then completed the COVID-19 questionnaire online. A subset of the new questionnaire respondents were invited to participate in COVID-19 serology substudies, using home antibody testing kits. Results: In total, 3,693 participants provided COVID-19 infection status (median age 62.9 (IQR 54.7-69.2), 58.9% female). Trends of monthly incidence proportions of COVID-19 in EXCEED (self-report or symptom-predicted) approximated local and national figures. Regression analysis of 2,768 participants with linked EHR data showed no obvious monotonic relationship between number of chronic diseases (of 16 pre-specified diseases) and COVID-19 infection. There were 2,144 participants with valid results from a kit allowing differentiation between antibodies due to vaccination or infection. Of these, 8.5% had results consistent with previous COVID-19 infection, and 85.9% had evidence of COVID-19 vaccination, but without evidence of infection. Conclusions: Enriching EXCEED with a new COVID-19 questionnaire and serology data may improve understanding of the risk factors, clinical sequelae and broader impacts of the COVID-19 pandemic in the general population. Controlled access to these data for bona fide researchers is via application to the EXCEED study.

Malignant Pleural Mesothelioma (MPM) is typically diagnosed 20-50 years after exposure to asbestos and evolves along an unknown evolutionary trajectory. To elucidate this path, we conducted multi-regional exome sequencing of 90 tumour samples from 22 MPMs acquired at surgery. Here we show that exomic intratumour heterogeneity varies widely across the cohort. Phylogenetic tree topology ranges from linear to highly branched, reflecting a steep gradient of genomic instability. Using transfer learning, we detect repeated evolution, resolving 5 clusters that are prognostic, with temporally ordered clonal drivers. BAP1/-3p21 and FBXW7/-chr4 events are always early clonal. In contrast, NF2/-22q events, leading to Hippo pathway inactivation are predominantly late clonal, positively selected, and when subclonal, exhibit parallel evolution indicating an evolutionary constraint. Very late somatic alteration of NF2/22q occurred in one patient 12 years after surgery. Clonal architecture and evolutionary clusters dictate MPM inflammation and immune evasion. These results reveal potentially drugable evolutionary bottlenecking in MPM, and an impact of clonal architecture on shaping the immune landscape, with potential to dictate the clinical response to immune checkpoint inhibition.

Common single-nucleotide variation in the host accounts for 25% of the variability in the plasma levels of HIV during the clinical latency stage (viral load set point). However, the role of rare variants and copy number variants remains relatively unexplored. Previous work has suggested copy number variation of a cluster of beta-defensin genes affects HIV load in treatment-na " ive sub-Saharan Africans and rate of response to antiretroviral treatment. Here we analyse a total of 1827 individuals from two cohorts of HIV-infected individuals from Europe and sub-Saharan Africa to investigate the role of beta-defensin copy number variation on HIV load at set point. We find no evidence for association of copy number with viral load. We also compare distribution of beta-defensin copy number between European cases and controls and find no differences, arguing against a role of beta-defensin copy number in HIV acquisition. Taken together, our data argue against an effect of copy number variation of the beta-defensin region in the spontaneous control of HIV infection.

EXCEED is a longitudinal population-based cohort which facilitates investigation of genetic, environmental and lifestyle-related determinants of a broad range of diseases and of multiple morbidity through data collected at baseline and via electronic healthcare record linkage. Recruitment has taken place in Leicester, Leicestershire and Rutland since 2013 and is ongoing, with 9,840 participants aged 30-69 to date. The population of Leicester is diverse and additional recruitment from the local South Asian community is ongoing. Participants have consented to follow-up for up to 25 years through electronic health records and additional bespoke data collection is planned. Data available includes baseline demographics, anthropometry, spirometry, lifestyle factors (smoking and alcohol use) and longitudinal health information from primary care records, with additional linkage to other EHR datasets planned. Patients have consented to be contacted for recall-by-genotype and recall-by-phenotype sub-studies, providing an important resource for precision medicine research. We welcome requests for collaboration and data access by contacting the study management team via exceed@le.ac.uk.

Glycophorin A and glycophorin B are red blood cell surface proteins that are both receptors for the parasitePlasmodium falciparum, which is the principal cause of malaria in sub-Saharan Africa. DUP4 is a complex structural genomic variant that carries extra copies of a glycophorin A - glycophorin B fusion gene, and has a dramatic effect on malaria risk by reducing the risk of severe malaria by up to 40%. Using fiber-FISH and Illumina sequencing, we validate the structural arrangement of the glycophorin locus in the DUP4 variant, and reveal somatic variation in copy number of the glycophorin A-glycophorin B fusion gene. By developing a simple, specific, PCR-based assay for DUP4 we show the DUP4 variant reaches a frequency of 13% in a village in south-eastern Tanzania. We genotype a substantial proportion of that village and demonstrate an association of DUP4 genotype with hemoglobin levels, a phenotype related to malaria, using a family-based association test. Taken together, we show that DUP4 is a complex structural variant that may be susceptible to somatic variation, and show that it is associated with a malarial-related phenotype in a non-hospitalized population.Significance statementPrevious work has identified a human complex genomic structural variant called DUP4, which includes two novel glycophorin A-glycophorin B fusion genes, is associated with a profound protection against severe malaria. In this study, we present data showing the molecular basis of this complex variant. We also show evidence of somatic variation in the copy number of the fusion genes. We develop a simple robust assay for this variant and demonstrate that DUP4 is at an appreciable population frequency in Tanzania and that it is associated with higher hemoglobin levels in a malaria-endemic village. We suggest that DUP4 is therefore protective against malarial anemia.

Background: The CCL3L1-CCR5 signaling axis is important in a number of inflammatory responses, including macrophage function, and T-cell-dependent immune responses. Small molecule CCR5 antagonists exist, including the approved antiretroviral drug maraviroc, and therapeutic monoclonal antibodies are in development. Repositioning of drugs and targets into new disease areas can accelerate the availability of new therapies and substantially reduce costs. As it has been shown that drug targets with genetic evidence supporting their involvement in the disease are more likely to be successful in clinical development, using genetic association studies to identify new target repurposing opportunities could be fruitful. Here we investigate the potential of perturbation of the CCL3L1-CCR5 axis as treatment for respiratory disease. Europeans typically carry between 0 and 5 copies of CCL3L1 and this multi-allelic variation is not detected by widely used genome-wide single nucleotide polymorphism studies. Methods: We directly measured the complex structural variation of CCL3L1 using the Paralogue Ratio Test and imputed (with validation) CCR5d32 genotypes in 5,000 individuals from UK Biobank, selected from the extremes of the lung function distribution, and analysed DNA and RNAseq data for CCL3L1 from the 1000 Genomes Project. Results: We confirmed the gene dosage effect of CCL3L1 copy number on CCL3L1 mRNA expression levels. We found no evidence for association of CCL3L1 copy number or CCR5d32 genotype with lung function. Conclusions: These results suggest that repositioning CCR5 antagonists is unlikely to be successful for the treatment of airflow obstruction.

Fcγ receptors (FcγRs) are key immune receptors responsible for the effective control of both humoral and innate immunity and are central to maintaining the balance between generating appropriate responses to infection and preventing autoimmunity. When this balance is lost, pathology results in increased susceptibility to cancer, autoimmunity, and infection. In contrast, optimal FcγR engagement facilitates effective disease resolution and response to monoclonal antibody immunotherapy. The underlying genetics of the FcγR gene family are a central component of this careful balance. Complex in humans and generated through ancestral duplication events, here we review the evolution of the gene family in mammals, the potential importance of copy number, and functionally relevant single nucleotide polymorphisms, as well as discussing current approaches and limitations when exploring genetic variation in this region.

Human beta defensin 2 (HBD2) is an endogenous mucosal antimicrobial peptide (AMP) upregulated during infection and inflammation. HBD2 is encoded by the DEFB4 gene, which exhibits extensive copy number variation. Previous studies have demonstrated a relationship between HBD copy number and serum HBD2 protein levels; however, our current understanding of the influence of copy number on mucosal AMP function remains limited. This study explores the relationship between HBD copy number, cervicovaginal HBD2 protein levels and antimicrobial activity in 203 women with risk factors for preterm birth. We provide evidence that suggests HBD copy number modulates cervical antimicrobial immunity.