Edward J. Hollox

Exposure to asbestos is the primary cause of mesothelioma, however the processes underpinning its initiation and evolutionary trajectory remain elusive. To address this knowledge gap, we conducted whole exome sequencing and phylogenetic analysis of 277 mesotheliomas. Using multidimensional Dirichlet process clustering we inferred phylogenies, uncovering mutation order dominated by early 13q,1p, 8p, 6q and 22q somatic copy number alterations. Mesotheliomas exhibit a spectrum of genomic intra-tumor heterogeneity spanning linear (L) to highly branched (B) tree topologies, quantifiable by the Weighted Shannon Entropy (WSE). Somatic copy number alterations emerge earlier than single nucleotide variants (SNVs), and at a much faster relative rate in L-mesotheliomas. BAP1 is the only positively selected driver in L-mesotheliomas, whereas, B-mesotheliomas harbor multiple positively selected drivers and an aggressive phenotype, including a bias of loss of RPL5, and MTAP deletion linked to breakpoint enriched with long interspersed nuclear elements. L-trees had lower WSE, but greater T-cell inflammation and tertiary lymphoid structures, despite having a lower neoantigen burden. WSE is higher in larger volume, immunologically cold mesotheliomas, and is associated with shorter survival. In summary, progressive genomic heterogeneity is a defining feature of more aggressive mesothelioma yet illuminates opportunities for precision therapeutic intervention.

The contribution of multi-allelic CNVs (mCNVs) to disease risk has not been widely studied. This is largely because they have been difficult to characterise at a large-scale genome-wide, and are often not strongly associated with flanking SNVs, limiting imputation. Improved understanding of the role of mCNVs in disease risk could lead to novel insights into the pathobiology of disease. We robustly typed 69 mCNVs from UK Biobank whole exome sequences in discovery (n=150,682) and replication sets (n=269,317). Discovery and replication PheWAS used clinically-curated composite phenotypes by integrating self-report, primary and secondary health care data to interrogate these variants, for unrelated British individuals of African, European and Central/South Asian ancestries. 173 mCNV-phenotype associations were detected from 26 mCNVs, of which 114 associations replicated. One of eight potentially novel mCNV-phenotype signals was independent of neighbouring associated SNVs, the association of Sulfotransferase 1A1 and 1A2 genes ( SULT1A1 / SULT1A2) with estimated glomerular filtration rate (eGFR) in individuals of European ancestry (meta-analysed p=1.05×10 ^-9 , beta=0.016 [0.011; 0.021]). Other potentially novel associations include Golgi phosphoprotein 3 ( GOLPH3) with the cardiovascular phenotype bundle branch block in individuals of South Asian ancestry (meta-analysed p=3.35×10 ^-6 , OR=2.13 [1.53, 2.96]) and alpha amylase 2B ( AMY2B ) with ventricular fibrillation and flutter in individuals of European ancestry (meta-analysed p=2.48×10 ^-6 , OR=1.50 [1.26; 1.78]). In summary, we show that accurate typing of biobank-scale sample sizes can identify associations between traits and mCNVs, acting through a gene dosage relationship. Our work provides several novel likely causative variants contributing to particular traits of clinical importance and immediately suggest a putative functional mechanism for the observed associations.

Malignant pleural mesothelioma (MPM) is a rare malignancy characterised by extensive structural genomic alterations and a low burden of recurrent single nucleotide variants. However, the full spectrum and functional impact of structural variation (SVs) remain incompletely understood because short-read sequencing has limitation ability to resolve complex genomic rearrangements. Here, we performed integrated short-read and long-read whole-genome sequencing on tumour-normal pairs from three MPM patients, together with RNA sequencing and nanopore-derived promoter methylation profiling. Long-reads sequencing substantially improved SV detection, identifying 61-156 novel SVs per sample, including complex rearrangements and breakpoint-resolved events affecting cancer-associated genes. Complex SV clusters consistent with chromoplexy and chromothripsis were observed and frequently involved oncogenes. Integration with transcriptomics data showed that several SVs-affected genes, including WEE1 and GPC6 , exhibited increased expression independent of gene dosage. Promoter methylation analysis revealed a conserved bimodal methylation landscape across tumours and a significant inverse relationship with gene expression. SV-associated genes showed coordinated promoter hypermethylation and transcriptional activation, suggesting that SVs may influence gene regulation through epigenetic mechanisms. Survival analysis using the TCGA-MESO cohort further showed that elevated expression of WEE1 and GPC6 was associated with poorer overall survival. Together, these findings highlight the value of long-read sequencing for uncovering functionally and clinically relevant structural variation in MPM.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease with a strong genetic component, yet a substantial proportion of its genetic risk remains unexplained by single-nucleotide variant (SNV) association studies. In this study, we conduct a genome-wide association study of tandem repeats (TRs), which are a pervasive and understudied class of genetic variation, to identify novel loci influencing IPF susceptibility. Using whole-genome sequencing data from two case-control datasets (discovery: 507 cases and 174 controls; replication: 1243 cases and 3197 controls), we identify and replicate associations at four TR loci, most notably a complex repeat within intron 2 of SLC15A4 , a gene encoding the endolysosomal peptide-histidine transporter 1 (PHT1). This TR is within a predicted enhancer and acts as an expression and splicing quantitative trait locus (eQTL/sQTL), influencing the expression of an alternative SLC15A4 transcript in biologically-relevant tissues and cells, with longer TR alleles correlating with decreased expression of this alternative transcript and increased IPF risk. In a biologically-relevant cell-line model, we show that this alternative transcript is targeted by nonsense-mediated mRNA decay, and levels of the alternative transcript are inversely correlated with levels of the canonical full-length SLC15A4 transcript. Our findings implicate SLC15A4 in IPF pathogenesis, possibly via modulation of innate immune responses to injury, and demonstrate the power of TR-focused genomic analyses to reveal previously undetectable disease mechanisms and therapeutic targets.

Leveraging adaptive tumour immunity to control mesothelioma via immune checkpoint blockade is now a standard therapeutic approach. However, the determinants of sensitivity remain elusive. Low non-synonymous mutation burden and programmed death-ligand 1 expression, an abundance of immunosuppressive immune cell infiltration, and 9p21 deletion should all mitigate responses to therapy. To address this knowledge gap, we conducted a double blind, placebo-controlled, randomized phase III trial of the PD1 inhibitor, nivolumab (ClinicalTrial.gov registration: NCT03063450). After 37.2 months of follow-up, the primary endpoint of progression free-survival, but not overall survival was met. The nivolumab response rate was 10.3%, and related grade 3 or above adverse events occurred in 20.4% versus 7.2% for placebo. Progression-free and overall survival were longer in nivolumab-treated responders versus non-responders. In an exploratory multiomic analysis, blinded whole exome, transcriptome and multiplex immune profiling were used to interrogate R- versus NR-subgroups. Non-synonymous and neoantigen mutation burden were no different between groups, however R-mesotheliomas were infiltrated with activated CD8⁺ T- and CD19⁺ B-lymphocytes, organised into tertiary lymphoid structures. B-cell infiltration correlated with pro-inflammatory chemokines including IL24 and CCL19. Conversely, epithelial-mesenchymal transition and mitosis were associated with resistance to nivolumab. These findings illuminate features which can be leveraged to advance precision immunotherapy in this rare cancer setting.

Background: Chronic dry cough is a symptom of common lung conditions, can occur as a side-effect of angiotensin-converting enzyme inhibitors (ACEis), or may be unexplained. Despite the substantial health burden presented by chronic dry cough, its biological mechanisms remain unclear. We hypothesised shared genetic architecture between chronic dry cough and ACEi-induced cough and aimed to identify causal genes underlying both phenotypes. Methods: We performed multi-ancestry genome-wide association studies (GWAS) of chronic dry cough and ACEi-induced cough, and a multi-trait GWAS of both phenotypes, utilising data from five cohort studies. Chronic dry cough was defined by questionnaire responses, and ACEi-induced cough by treatment switches or clinical diagnosis in electronic health records. We mapped putative causal genes and performed phenome-wide association studies (PheWAS) of associated variants, and polygenic scores for ACEi-induced cough, to identify pleiotropic effects. Results: We found seven novel genetic association signals reaching p–8 in the multi-trait or single-trait analyses of chronic dry cough and ACEi-induced cough. The novel variants mapped to 10 novel genes, and we mapped an additional three novel genes to known risk variants, many of which implicate neurological functions ( CTNNA1 , KCNA10 , MAPKAP1 , OR4C12 , OR4C13 , SIL1 ). The polygenic-score-based PheWAS highlighted associations with an elevated risk of several clinical conditions including asthma, diabetes and multi-site chronic pain. Conclusion: Our findings provide support for neuronal dysfunction underlying cough hypersensitivity in chronic dry cough and ACEi-induced cough, and identify diseases and traits associated with genetic predisposition to cough that could inform drug target discovery.

β-Defensins are peptides with antimicrobial roles, characterized by a conserved tertiary structure. Beyond antimicrobial functions, they exhibit diverse roles in both the immune response and fertility, including involvement in sperm maturation and function. Copy number variation (CNV) of β-defensin genes is extensive across mammals, including cattle, with possible implications for reproductive traits and disease resistance. In this study, we comprehensively catalogue 55 β-defensin genes in cattle. By constructing a phylogenetic tree to identify human orthologues and lineage-specific expansions, we identify 1 : 1 human orthologues for 35 bovine β-defensins. We also discover extensive β-defensin gene CNV across breeds, with DEFB103, in particular, showing extensive multi-allelic CNV. By comparing β-defensin expression levels in testis from calves and adult bulls, we find that 14 β-defensins, including DEFB103 , increase in expression during sexual maturation. Analysis of β-defensin gene expression levels in the caput of adult bull epididymis, and β-defensin gene copy number, in 94 matched samples shows expression levels of four β-defensins are correlated with genomic copy numbers, including DEFB103 . We therefore demonstrate extensive CNV in bovine β-defensin genes, in particular DEFB103 , with potential functional consequences for fertility.

Decades of genetic association testing in human cohorts have provided important insights into the genetic architecture and biological underpinnings of complex traits and diseases. However, for certain traits, genome-wide association studies (GWAS) for common SNPs are approaching signal saturation, which underscores the need to explore other types of genetic variation to understand the genetic basis of traits and diseases. Copy number variation (CNV) is an important source of heritability that is well known to functionally affect human traits. Recent technological and computational advances enable the large-scale, genome-wide evaluation of CNVs, with implications for downstream applications such as polygenic risk scoring and drug target identification. Here, we review the current state of CNV-GWAS, discuss current limitations in resource infrastructure that need to be overcome to enable the wider uptake of CNV-GWAS results, highlight emerging opportunities and suggest guidelines and standards for future GWAS for genetic variation beyond SNPs at scale.

Malignant mesothelioma is a rare tumour caused by asbestos exposure that originates mainly from the pleural lining or the peritoneum. Treatment options are limited, and the prognosis is dismal. Although immune checkpoint blockade (ICB) can improve survival outcomes, the determinants of responsiveness remain elusive. Here, we report the outcomes of a multi-centre phase II clinical trial (MiST4, NCT03654833) evaluating atezolizumab and bevacizumab (AtzBev) in patients with relapsed mesothelioma. We also use tumour tissue and gut microbiome sequencing, as well as tumour spatial immunophenotyping to identify factors associated with treatment response. MIST4 met its primary endpoint with 50% 12-week disease control, and the treatment was tolerable. Aneuploidy, notably uniparental disomy (UPD), homologous recombination deficiency (HRD), epithelial-mesenchymal transition and inflammation with CD68⁺ monocytes were identified as tumour-intrinsic resistance factors. The log-ratio of gut-resident microbial genera positively correlated with radiological response to AtzBev and CD8⁺ T cell infiltration, but was inversely correlated with UPD, HRD and tumour infiltration by CD68⁺ monocytes. In summary, a model is proposed in which both intrinsic and extrinsic determinants in mesothelioma cooperate to modify the tumour microenvironment and confer clinical sensitivity to AtzBev. Gut microbiota represent a potentially modifiable factor with potential to improve immunotherapy outcomes for individuals with this cancer of unmet need.

β-defensins are peptides with antimicrobial roles, characterized by a conserved tertiary structure. Beyond antimicrobial functions, they exhibit diverse roles in both the immune response and fertility, including involvement in sperm maturation and function. Copy number variation (CNV) of β-defensin genes is extensive across mammals, including cattle, with possible implications for reproductive traits and disease resistance. In this study, we comprehensively catalogue 55 β-defensin genes in cattle. By constructing a phylogenetic tree to identify human orthologues and lineage-specific expansions, we identify 1:1 human orthologues for 35 bovine β-defensins. We also discover extensive β-defensin gene CNV across breeds, with DEFB103 in particular showing extensive multiallelic CNV. By comparing β-defensin expression levels in testis from calves and adult bulls, we find that 14 β-defensins, including DEFB103 , increase in expression during sexual maturation. Analysis of β-defensin gene expression levels in the caput of adult bull epididymis, and β-defensin gene copy number, in 94 matched samples shows expression level of four β-defensins are correlated with genomic copy number, including DEFB103 . We therefore demonstrate extensive copy number variation in bovine β-defensin genes, in particular DEFB103 , with potential functional consequences for fertility.