Large-scale analysis of whole genome sequencing data from formalin-fixed paraffin-embedded cancer specimens demonstrates preservation of clinical utility.

Whole genome sequencing (WGS) provides comprehensive, individualised cancer genomic information. However, routine tumour biopsies are formalin-fixed and paraffin-embedded (FFPE), damaging DNA, historically limiting their use in WGS. Here we analyse FFPE cancer WGS datasets from England's 100,000 Genomes Project, comparing 578 FFPE samples with 11,014 fresh frozen (FF) samples across multiple tumour types. We use an approach that characterises rather than discards artefacts. We identify three artefactual signatures, including one known (SBS57) and two previously uncharacterised (SBS FFPE, ID FFPE), and develop an "FFPEImpact" score that quantifies sample artefacts. Despite inferior sequencing quality, FFPE-derived data identifies clinically-actionable variants, mutational signatures and permits algorithmic stratification. Matched FF/FFPE validation cohorts shows good concordance while acknowledging SBS, ID and copy-number artefacts. While FF-derived WGS data remains the gold standard, FFPE-samples can be used for WGS if required, using analytical advancements developed here, potentially democratising whole cancer genomics to many.

A Comparison of Structural Variant Calling from Short-Read and Nanopore-Based Whole-Genome Sequencing Using Optical Genome Mapping as a Benchmark.

The identification of structural variants (SVs) in genomic data represents an ongoing challenge because of difficulties in reliable SV calling leading to reduced sensitivity and specificity. We prepared high-quality DNA from 9 parent-child trios, who had previously undergone short-read whole-genome sequencing (Illumina platform) as part of the Genomics England 100,000 Genomes Project. We reanalysed the genomes using both Bionano optical genome mapping (OGM; 8 probands and one trio) and Nanopore long-read sequencing (Oxford Nanopore Technologies [ONT] platform; all samples). To establish a "truth" dataset, we asked whether rare proband SV calls (n = 234) made by the Bionano Access (version 1.6.1)/Solve software (version 3.6.1_11162020) could be verified by individual visualisation using the Integrative Genomics Viewer with either or both of the Illumina and ONT raw sequence. Of these, 222 calls were verified, indicating that Bionano OGM calls have high precision (positive predictive value 95%). We then asked what proportion of the 222 true Bionano SVs had been identified by SV callers in the other two datasets. In the Illumina dataset, sensitivity varied according to variant type, being high for deletions (115/134; 86%) but poor for insertions (13/58; 22%). In the ONT dataset, sensitivity was generally poor using the original Sniffles variant caller (48% overall) but improved substantially with use of Sniffles2 (36/40; 90% and 17/23; 74% for deletions and insertions, respectively). In summary, we show that the precision of OGM is very high. In addition, when applying the Sniffles2 caller, the sensitivity of SV calling using ONT long-read sequence data outperforms Illumina sequencing for most SV types.

Long read sequencing characterises a novel structural variant, revealing underactive AKR1C1 with overactive AKR1C2 as a possible cause of severe chronic fatigue.

BACKGROUND: Causative genetic variants cannot yet be found for many disorders with a clear heritable component, including chronic fatigue disorders like myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). These conditions may involve genes in difficult-to-align genomic regions that are refractory to short read approaches. Structural variants in these regions can be particularly hard to detect or define with short reads, yet may account for a significant number of cases. Long read sequencing can overcome these difficulties but so far little data is available regarding the specific analytical challenges inherent in such regions, which need to be taken into account to ensure that variants are correctly identified. Research into chronic fatigue disorders faces the additional challenge that the heterogeneous patient populations likely encompass multiple aetiologies with overlapping symptoms, rather than a single disease entity, such that each individual abnormality may lack statistical significance within a larger sample. Better delineation of patient subgroups is needed to target research and treatment. METHODS: We use nanopore sequencing in a case of unexplained severe fatigue to identify and fully characterise a large inversion in a highly homologous region spanning the AKR1C gene locus, which was indicated but could not be resolved by short-read sequencing. We then use GC-MS/MS serum steroid analysis to investigate the functional consequences. RESULTS: Several commonly used bioinformatics tools are confounded by the homology but a combined approach including visual inspection allows the variant to be accurately resolved. The DNA inversion appears to increase the expression of AKR1C2 while limiting AKR1C1 activity, resulting in a relative increase of inhibitory GABAergic neurosteroids and impaired progesterone metabolism which could suppress neuronal activity and interfere with cellular function in a wide range of tissues. CONCLUSIONS: This study provides an example of how long read sequencing can improve diagnostic yield in research and clinical care, and highlights some of the analytical challenges presented by regions containing tandem arrays of genes. It also proposes a novel gene associated with a novel disease aetiology that may be an underlying cause of complex chronic fatigue. It reveals biomarkers that could now be assessed in a larger cohort, potentially identifying a subset of patients who might respond to treatments suggested by the aetiology.

Nuclear-embedded mitochondrial DNA sequences in 66,083 human genomes.

DNA transfer from cytoplasmic organelles to the cell nucleus is a legacy of the endosymbiotic event-the majority of nuclear-mitochondrial segments (NUMTs) are thought to be ancient, preceding human speciation1-3. Here we analyse whole-genome sequences from 66,083 people-including 12,509 people with cancer-and demonstrate the ongoing transfer of mitochondrial DNA into the nucleus, contributing to a complex NUMT landscape. More than 99% of individuals had at least one of 1,637 different NUMTs, with 1 in 8 individuals having an ultra-rare NUMT that is present in less than 0.1% of the population. More than 90% of the extant NUMTs that we evaluated inserted into the nuclear genome after humans diverged from apes. Once embedded, the sequences were no longer under the evolutionary constraint seen within the mitochondrion, and NUMT-specific mutations had a different mutational signature to mitochondrial DNA. De novo NUMTs were observed in the germline once in every 104 births and once in every 103 cancers. NUMTs preferentially involved non-coding mitochondrial DNA, linking transcription and replication to their origin, with nuclear insertion involving multiple mechanisms including double-strand break repair associated with PR domain zinc-finger protein 9 (PRDM9) binding. The frequency of tumour-specific NUMTs differed between cancers, including a probably causal insertion in a myxoid liposarcoma. We found evidence of selection against NUMTs on the basis of size and genomic location, shaping a highly heterogenous and dynamic human NUMT landscape.

Whole-genome sequencing reveals host factors underlying critical COVID-19.

Critical COVID-19 is caused by immune-mediated inflammatory lung injury. Host genetic variation influences the development of illness requiring critical care1 or hospitalization2-4 after infection with SARS-CoV-2. The GenOMICC (Genetics of Mortality in Critical Care) study enables the comparison of genomes from individuals who are critically ill with those of population controls to find underlying disease mechanisms. Here we use whole-genome sequencing in 7,491 critically ill individuals compared with 48,400 controls to discover and replicate 23 independent variants that significantly predispose to critical COVID-19. We identify 16 new independent associations, including variants within genes that are involved in interferon signalling (IL10RB and PLSCR1), leucocyte differentiation (BCL11A) and blood-type antigen secretor status (FUT2). Using transcriptome-wide association and colocalization to infer the effect of gene expression on disease severity, we find evidence that implicates multiple genes-including reduced expression of a membrane flippase (ATP11A), and increased expression of a mucin (MUC1)-in critical disease. Mendelian randomization provides evidence in support of causal roles for myeloid cell adhesion molecules (SELE, ICAM5 and CD209) and the coagulation factor F8, all of which are potentially druggable targets. Our results are broadly consistent with a multi-component model of COVID-19 pathophysiology, in which at least two distinct mechanisms can predispose to life-threatening disease: failure to control viral replication; or an enhanced tendency towards pulmonary inflammation and intravascular coagulation. We show that comparison between cases of critical illness and population controls is highly efficient for the detection of therapeutically relevant mechanisms of disease.

Inherited duplications of PPP2R3B predispose to nevi and melanoma via a C21orf91-driven proliferative phenotype.

PURPOSE: Much of the heredity of melanoma remains unexplained. We sought predisposing germline copy-number variants using a rare disease approach. METHODS: Whole-genome copy-number findings in patients with melanoma predisposition syndrome congenital melanocytic nevus were extrapolated to a sporadic melanoma cohort. Functional effects of duplications in PPP2R3B were investigated using immunohistochemistry, transcriptomics, and stable inducible cellular models, themselves characterized using RNAseq, quantitative real-time polymerase chain reaction (qRT-PCR), reverse phase protein arrays, immunoblotting, RNA interference, immunocytochemistry, proliferation, and migration assays. RESULTS: We identify here a previously unreported genetic susceptibility to melanoma and melanocytic nevi, familial duplications of gene PPP2R3B. This encodes PR70, a regulatory unit of critical phosphatase PP2A. Duplications increase expression of PR70 in human nevus, and increased expression in melanoma tissue correlates with survival via a nonimmunological mechanism. PPP2R3B overexpression induces pigment cell switching toward proliferation and away from migration. Importantly, this is independent of the known microphthalmia-associated transcription factor (MITF)-controlled switch, instead driven by C21orf91. Finally, C21orf91 is demonstrated to be downstream of MITF as well as PR70. CONCLUSION: This work confirms the power of a rare disease approach, identifying a previously unreported copy-number change predisposing to melanocytic neoplasia, and discovers C21orf91 as a potentially targetable hub in the control of phenotype switching.

An ancestral 10-bp repeat expansion in VWA1 causes recessive hereditary motor neuropathy.

The extracellular matrix comprises a network of macromolecules such as collagens, proteoglycans and glycoproteins. VWA1 (von Willebrand factor A domain containing 1) encodes a component of the extracellular matrix that interacts with perlecan/collagen VI, appears to be involved in stabilizing extracellular matrix structures, and demonstrates high expression levels in tibial nerve. Vwa1-deficient mice manifest with abnormal peripheral nerve structure/function; however, VWA1 variants have not previously been associated with human disease. By interrogating the genome sequences of 74 180 individuals from the 100K Genomes Project in combination with international gene-matching efforts and targeted sequencing, we identified 17 individuals from 15 families with an autosomal-recessive, non-length dependent, hereditary motor neuropathy and rare biallelic variants in VWA1. A single disease-associated allele p.(G25Rfs*74), a 10-bp repeat expansion, was observed in 14/15 families and was homozygous in 10/15. Given an allele frequency in European populations approaching 1/1000, the seven unrelated homozygote individuals ascertained from the 100K Genomes Project represents a substantial enrichment above expected. Haplotype analysis identified a shared 220 kb region suggesting that this founder mutation arose >7000 years ago. A wide age-range of patients (6-83 years) helped delineate the clinical phenotype over time. The commonest disease presentation in the cohort was an early-onset (mean 2.0 ± 1.4 years) non-length-dependent axonal hereditary motor neuropathy, confirmed on electrophysiology, which will have to be differentiated from other predominantly or pure motor neuropathies and neuronopathies. Because of slow disease progression, ambulation was largely preserved. Neurophysiology, muscle histopathology, and muscle MRI findings typically revealed clear neurogenic changes with single isolated cases displaying additional myopathic process. We speculate that a few findings of myopathic changes might be secondary to chronic denervation rather than indicating an additional myopathic disease process. Duplex reverse transcription polymerase chain reaction and immunoblotting using patient fibroblasts revealed that the founder allele results in partial nonsense mediated decay and an absence of detectable protein. CRISPR and morpholino vwa1 modelling in zebrafish demonstrated reductions in motor neuron axonal growth, synaptic formation in the skeletal muscles and locomotive behaviour. In summary, we estimate that biallelic variants in VWA1 may be responsible for up to 1% of unexplained hereditary motor neuropathy cases in Europeans. The detailed clinical characterization provided here will facilitate targeted testing on suitable patient cohorts. This novel disease gene may have previously evaded detection because of high GC content, consequential low coverage and computational difficulties associated with robustly detecting repeat-expansions. Reviewing previously unsolved exomes using lower QC filters may generate further diagnoses.

Publisher Correction: c-Maf controls immune responses by regulating disease-specific gene networks and repressing IL-2 in CD4+ T cells.

In the version of this article initially published, the Supplementary Data file was an incorrect version. The correct version is now provided. The error has been corrected in the HTML and PDF version of the article.

Low plasma haptoglobin is a risk factor for life-threatening childhood severe malarial anemia and not an exclusive consequence of hemolysis.

Severe Malarial Anemia (SMA), a life-threatening childhood Plasmodium falciparum malaria syndrome requiring urgent blood transfusion, exhibits inflammatory and hemolytic pathology. Differentiating between hypo-haptoglobinemia due to hemolysis or that of genetic origin is key to understand SMA pathogenesis. We hypothesized that while malaria-induced hypo-haptoglobinemia should reverse at recovery, that of genetic etiology should not. We carried-out a case-control study of children living under hyper-endemic holoendemic malaria burden in the sub-Saharan metropolis of Ibadan, Nigeria. We show that hypo-haptoglobinemia is a risk factor for childhood SMA and not solely due to intravascular hemolysis from underlying schizogony. In children presenting with SMA, hypo-haptoglobinemia remains through convalescence to recovery suggesting a genetic cause. We identified a haptoglobin gene variant, rs12162087 (g.-1203G > A, frequency = 0.67), to be associated with plasma haptoglobin levels (p = 8.5 × 10-6). The Homo-Var:(AA) is associated with high plasma haptoglobin while the reference Homo-Ref:(GG) is associated with hypo-haptoglobinemia (p = 2.3 × 10-6). The variant is associated with SMA, with the most support for a risk effect for Homo-Ref genotype. Our insights on regulatory haptoglobin genotypes and hypo-haptoglobinemia suggest that haptoglobin screening could be part of risk-assessment algorithms to prevent rapid disease progression towards SMA in regions with no-access to urgent blood transfusion where SMA accounts for high childhood mortality rates.

Publisher Correction: The sea lamprey germline genome provides insights into programmed genome rearrangement and vertebrate evolution.

When published, this article did not initially appear open access. This error has been corrected, and the open access status of the paper is noted in all versions of the paper. Additionally, affiliation 16 denoting equal contribution was missing from author Robb Krumlauf in the PDF originally published. This error has also been corrected.

Deterministic Evolutionary Trajectories Influence Primary Tumor Growth: TRACERx Renal.

The evolutionary features of clear-cell renal cell carcinoma (ccRCC) have not been systematically studied to date. We analyzed 1,206 primary tumor regions from 101 patients recruited into the multi-center prospective study, TRACERx Renal. We observe up to 30 driver events per tumor and show that subclonal diversification is associated with known prognostic parameters. By resolving the patterns of driver event ordering, co-occurrence, and mutual exclusivity at clone level, we show the deterministic nature of clonal evolution. ccRCC can be grouped into seven evolutionary subtypes, ranging from tumors characterized by early fixation of multiple mutational and copy number drivers and rapid metastases to highly branched tumors with >10 subclonal drivers and extensive parallel evolution associated with attenuated progression. We identify genetic diversity and chromosomal complexity as determinants of patient outcome. Our insights reconcile the variable clinical behavior of ccRCC and suggest evolutionary potential as a biomarker for both intervention and surveillance.

Publisher Correction: The sea lamprey germline genome provides insights into programmed genome rearrangement and vertebrate evolution.

In the version of this article initially published, the present addresses for authors Dorit Hockman and Chris Amemiya were switched. The error has been corrected in the HTML and PDF versions of the article.

c-Maf controls immune responses by regulating disease-specific gene networks and repressing IL-2 in CD4+ T cells.

The transcription factor c-Maf induces the anti-inflammatory cytokine IL-10 in CD4+ T cells in vitro. However, the global effects of c-Maf on diverse immune responses in vivo are unknown. Here we found that c-Maf regulated IL-10 production in CD4+ T cells in disease models involving the TH1 subset of helper T cells (malaria), TH2 cells (allergy) and TH17 cells (autoimmunity) in vivo. Although mice with c-Maf deficiency targeted to T cells showed greater pathology in TH1 and TH2 responses, TH17 cell-mediated pathology was reduced in this context, with an accompanying decrease in TH17 cells and increase in Foxp3+ regulatory T cells. Bivariate genomic footprinting elucidated the c-Maf transcription-factor network, including enhanced activity of NFAT; this led to the identification and validation of c-Maf as a negative regulator of IL-2. The decreased expression of the gene encoding the transcription factor RORγt (Rorc) that resulted from c-Maf deficiency was dependent on IL-2, which explained the in vivo observations. Thus, c-Maf is a positive and negative regulator of the expression of cytokine-encoding genes, with context-specific effects that allow each immune response to occur in a controlled yet effective manner.

The sea lamprey germline genome provides insights into programmed genome rearrangement and vertebrate evolution.

The sea lamprey (Petromyzon marinus) serves as a comparative model for reconstructing vertebrate evolution. To enable more informed analyses, we developed a new assembly of the lamprey germline genome that integrates several complementary data sets. Analysis of this highly contiguous (chromosome-scale) assembly shows that both chromosomal and whole-genome duplications have played significant roles in the evolution of ancestral vertebrate and lamprey genomes, including chromosomes that carry the six lamprey HOX clusters. The assembly also contains several hundred genes that are reproducibly eliminated from somatic cells during early development in lamprey. Comparative analyses show that gnathostome (mouse) homologs of these genes are frequently marked by polycomb repressive complexes (PRCs) in embryonic stem cells, suggesting overlaps in the regulatory logic of somatic DNA elimination and bivalent states that are regulated by early embryonic PRCs. This new assembly will enhance diverse studies that are informed by lampreys' unique biology and evolutionary/comparative perspective.

Aberrant ribonucleotide incorporation and multiple deletions in mitochondrial DNA of the murine MPV17 disease model.

All DNA polymerases misincorporate ribonucleotides despite their preference for deoxyribonucleotides, and analysis of cultured cells indicates that mammalian mitochondrial DNA (mtDNA) tolerates such replication errors. However, it is not clear to what extent misincorporation occurs in tissues, or whether this plays a role in human disease. Here, we show that mtDNA of solid tissues contains many more embedded ribonucleotides than that of cultured cells, consistent with the high ratio of ribonucleotide to deoxynucleotide triphosphates in tissues, and that riboadenosines account for three-quarters of them. The pattern of embedded ribonucleotides changes in a mouse model of Mpv17 deficiency, which displays a marked increase in rGMPs in mtDNA. However, while the mitochondrial dGTP is low in the Mpv17-/- liver, the brain shows no change in the overall dGTP pool, leading us to suggest that Mpv17 determines the local concentration or quality of dGTP. Embedded rGMPs are expected to distort the mtDNA and impede its replication, and elevated rGMP incorporation is associated with early-onset mtDNA depletion in liver and late-onset multiple deletions in brain of Mpv17-/- mice. These findings suggest aberrant ribonucleotide incorporation is a primary mtDNA abnormality that can result in pathology.

MPV17 Loss Causes Deoxynucleotide Insufficiency and Slow DNA Replication in Mitochondria.

MPV17 is a mitochondrial inner membrane protein whose dysfunction causes mitochondrial DNA abnormalities and disease by an unknown mechanism. Perturbations of deoxynucleoside triphosphate (dNTP) pools are a recognized cause of mitochondrial genomic instability; therefore, we determined DNA copy number and dNTP levels in mitochondria of two models of MPV17 deficiency. In Mpv17 ablated mice, liver mitochondria showed substantial decreases in the levels of dGTP and dTTP and severe mitochondrial DNA depletion, whereas the dNTP pool was not significantly altered in kidney and brain mitochondria that had near normal levels of DNA. The shortage of mitochondrial dNTPs in Mpv17-/- liver slows the DNA replication in the organelle, as evidenced by the elevated level of replication intermediates. Quiescent fibroblasts of MPV17-mutant patients recapitulate key features of the primary affected tissue of the Mpv17-/- mice, displaying virtual absence of the protein, decreased dNTP levels and mitochondrial DNA depletion. Notably, the mitochondrial DNA loss in the patients' quiescent fibroblasts was prevented and rescued by deoxynucleoside supplementation. Thus, our study establishes dNTP insufficiency in the mitochondria as the cause of mitochondrial DNA depletion in MPV17 deficiency, and identifies deoxynucleoside supplementation as a potential therapeutic strategy for MPV17-related disease. Moreover, changes in the expression of factors involved in mitochondrial deoxynucleotide homeostasis indicate a remodeling of nucleotide metabolism in MPV17 disease models, which suggests mitochondria lacking functional MPV17 have a restricted purine mitochondrial salvage pathway.

Complete re-sequencing of a 2Mb topological domain encompassing the FTO/IRXB genes identifies a novel obesity-associated region upstream of IRX5.

BACKGROUND: Association studies have identified a number of loci that contribute to an increased body mass index (BMI), the strongest of which is in the first intron of the FTO gene on human chromosome 16q12.2. However, this region is both non-coding and under strong linkage disequilibrium, making it recalcitrant to functional interpretation. Furthermore, the FTO gene is located within a complex cis-regulatory landscape defined by a topologically associated domain that includes the IRXB gene cluster, a trio of developmental regulators. Consequently, at least three genes in this interval have been implicated in the aetiology of obesity. METHODS: Here, we sequence a 2 Mb region encompassing the FTO, RPGRIP1L and IRXB cluster genes in 284 individuals from a well-characterised study group of Danish men containing extremely overweight young adults and controls. We further replicate our findings both in an expanded male cohort and an independent female study group. Finally, we compare our variant data with a previous study describing IRX3 and FTO interactions in this region. RESULTS: We obtain deep coverage across the entire region, allowing accurate and unequivocal determination of almost every single nucleotide polymorphism and short insertion/deletion. As well as confirming previous findings across the interval, we identify a further novel age-dependent association upstream of IRX5 that imposes a similar burden on BMI to the FTO locus. CONCLUSIONS: Our findings are consistent with the hypothesis that chromatin architectures play a role in regulating gene expression levels across topological domains while our targeted sequence approach represents a widely applicable methodology for high-resolution analysis of regional variation across candidate genomic loci.

Evolution of lineage-specific functions in ancient cis-regulatory modules.

Morphological evolution is driven both by coding sequence variation and by changes in regulatory sequences. However, how cis-regulatory modules (CRMs) evolve to generate entirely novel expression domains is largely unknown. Here, we reconstruct the evolutionary history of a lens enhancer located within a CRM that not only predates the lens, a vertebrate innovation, but bilaterian animals in general. Alignments of orthologous sequences from different deuterostomes sub-divide the CRM into a deeply conserved core and a more divergent flanking region. We demonstrate that all deuterostome flanking regions, including invertebrate sequences, activate gene expression in the zebrafish lens through the same ancient cluster of activator sites. However, levels of gene expression vary between species due to the presence of repressor motifs in flanking region and core. These repressor motifs are responsible for the relatively weak enhancer activity of tetrapod flanking regions. Ray-finned fish, however, have gained two additional lineage-specific activator motifs which in combination with the ancient cluster of activators and the core constitute a potent lens enhancer. The exploitation and modification of existing regulatory potential in flanking regions but not in the highly conserved core might represent a more general model for the emergence of novel regulatory functions in complex CRMs.

Identification and functional characterization of novel transcriptional enhancers involved in regulating human GLI3 expression during early development.

The zinc-finger transcription factor GLI3 acts as a primary transducer of Sonic hedgehog (Shh) signaling in a context-dependent combinatorial fashion. GLI3 participates in the patterning and growth of many organs, including the central nervous system (CNS) and limbs. Previously, we reported a subset of human intronic cis-regulators controlling many known aspects of endogenous Gli3 expression in mouse and zebrafish. Here we demonstrate in a transgenic zebrafish assay the potential of two novel tetrapod-teleost conserved non-coding elements (CNEs) docking within GLI3 intronic intervals (intron 3 and 4) to induce reporter gene expression at known sites of endogenous Gli3 transcription in embryonic domains such as the central nervous system (CNS) and limbs. Interestingly, the cell culture based assays reveal harmony with the context dependent dual nature of intra-GLI3 conserved elements. Furthermore, a transgenic zebrafish assay of previously reported limb-specific GLI3 transcriptional enhancers (previously tested in mice and chicken limb buds) induced reporter gene expression in zebrafish blood precursor cells and notochord instead of fin. These results demonstrate that the appendage-specific activity of a subset of GLI3-associated enhancers might be a tetrapod innovation. Taken together with our recent data, these results suggest that during the course of vertebrate evolution Gli3 expression control acquired a complex cis-regulatory landscape for spatiotemporal patterning of CNS and limbs. Comparative data from fish and mice suggest that the functional aspects of a subset of these cis-regulators have diverged significantly between these two lineages.