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.

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.

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.

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.

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.

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.

Persistence of duplicated PAC1 receptors in the teleost, Sparus auratus.

BACKGROUND: : Duplicated genes are common in vertebrate genomes. Their persistence is assumed to be either a consequence of gain of novel function (neofunctionalisation) or partitioning of the function of the ancestral molecule (sub-functionalisation). Surprisingly few studies have evaluated the extent of such modifications despite the numerous duplicated receptor and ligand genes identified in vertebrate genomes to date. In order to study the importance of function in the maintenance of duplicated genes, sea bream (Sparus auratus) PAC1 receptors, sequence homologues of the mammalian receptor specific for PACAP (Pituitary Adenylate Cyclase-Activating Polypeptide), were studied. These receptors belong to family 2 GPCRs and most of their members are duplicated in teleosts although the reason why both persist in the genome is unknown. RESULTS: : Duplicate sea bream PACAP receptor genes (sbPAC1A and sbPAC1B), members of family 2 GPCRs, were isolated and share 77% amino acid sequence identity. RT-PCR with specific primers for each gene revealed that they have a differential tissue distribution which overlaps with the distribution of the single mammalian receptor. Furthermore, in common with mammals, the teleost genes undergo alternative splicing and a PAC1Ahop1 isoform has been characterised. Duplicated orthologous receptors have also been identified in other teleost genomes and their distribution profile suggests that function may be species specific. Functional analysis of the paralogue sbPAC1s in Cos7 cells revealed that they are strongly stimulated in the presence of mammalian PACAP27 and PACAP38 and far less with VIP (Vasoactive Intestinal Peptide). The sbPAC1 receptors are equally stimulated (LOGEC50 values for maximal cAMP production) in the presence of PACAP27 (-8.74 +/- 0.29 M and -9.15 +/- 0.21 M, respectively for sbPAC1A and sbPAC1B, P > 0.05) and PACAP38 (-8.54 +/- 0.18 M and -8.92 +/- 0.24 M, respectively for sbPAC1A and sbPAC1B, P > 0.05). Human VIP was found to stimulate sbPAC1A (-7.23 +/- 0.20 M) more strongly than sbPAC1B (-6.57 +/- 0.14 M, P < 0.05) and human secretin (SCT), which has not so far been identified in fish genomes, caused negligible stimulation of both receptors. CONCLUSION: : The existence of functionally divergent duplicate sbPAC1 receptors is in line with previously proposed theories about the origin and maintenance of duplicated genes. Sea bream PAC1 duplicate receptors resemble the typical mammalian PAC1, and PACAP peptides were found to be more effective than VIP in stimulating cAMP production, although sbPAC1A was more responsive for VIP than sbPAC1B. These results together with the highly divergent pattern of tissue distribution suggest that a process involving neofunctionalisation occurred after receptor duplication within the fish lineage and probably accounts for their persistence in the genome. The characterisation of further duplicated receptors and their ligands should provide insights into the evolution and function of novel protein-protein interactions associated with the vertebrate radiation.

Ultraconserved non-coding sequence element controls a subset of spatiotemporal GLI3 expression.

The zinc-finger transcription factor GLI3 acts during vertebrate development in a combinatorial, context-dependent fashion as a primary transducer of sonic hedgehog (SHH) signaling. In humans, mutations affecting this key regulator of development are associated with GLI3-morphopathies, a group of congenital malformations in which forebrain and limb development are preferentially affected. We show that a non-coding element from intron two of GLI3, ultraconserved in mammals and highly conserved in the pufferfish Fugu, is a transcriptional enhancer. In transient transfection assays, it activates reporter gene transcription in human cell cultures expressing endogenous GLI3 but not in GLI3 negative cells. The identified enhancer element is predicted to contain conserved binding sites for transcription factors crucial for developmental steps in which GLI3 is involved. The regulatory potential of this element is conserved and was used to direct tissue-specific expression of a green fluorescent protein reporter gene in zebrafish embryos and of a beta-galactosidase reporter in transgenic mouse embryos. Time, location, and quantity of reporter gene expression are congruent with part of the pattern previously reported for endogenous GLI3 transcription.

Human GLI3 intragenic conserved non-coding sequences are tissue-specific enhancers.

The zinc-finger transcription factor GLI3 is a key regulator of development, acting as a primary transducer of Sonic hedgehog (SHH) signaling in a combinatorial context dependent fashion controlling multiple patterning steps in different tissues/organs. A tight temporal and spatial control of gene expression is indispensable, however, cis-acting sequence elements regulating GLI3 expression have not yet been reported. We show that 11 ancient genomic DNA signatures, conserved from the pufferfish Takifugu (Fugu) rubripes to man, are distributed throughout the introns of human GLI3. They map within larger conserved non-coding elements (CNEs) that are found in the tetrapod lineage. Full length CNEs transiently transfected into human cell cultures acted as cell type specific enhancers of gene transcription. The regulatory potential of these elements is conserved and was exploited to direct tissue specific expression of a reporter gene in zebrafish embryos. Assays of deletion constructs revealed that the human-Fugu conserved sequences within the GLI3 intronic CNEs were essential but not sufficient for full-scale transcriptional activation. The enhancer activity of the CNEs is determined by a combinatorial effect of a core sequence conserved between human and teleosts (Fugu) and flanking tetrapod-specific sequences, suggesting that successive clustering of sequences with regulatory potential around an ancient, highly conserved nucleus might be a possible mechanism for the evolution of cis-acting regulatory elements.

Different words, same meaning: understanding the languages of the genome.

There is undeniable value in using sequence comparison to identify putative regulatory sequences. However, a recent report has demonstrated that not all regulatory sequences are evolutionarily conserved. Cis-acting sequences around the RET gene, conserved in mammals but not in fish, are able to reproduce patterns of RET expression in zebrafish embryos. It is as yet unclear whether these sequences are 'below the radar' of current sequence alignment tools or whether their functional homology is not sequence based.

Highly conserved non-coding sequences are associated with vertebrate development.

In addition to protein coding sequence, the human genome contains a significant amount of regulatory DNA, the identification of which is proving somewhat recalcitrant to both in silico and functional methods. An approach that has been used with some success is comparative sequence analysis, whereby equivalent genomic regions from different organisms are compared in order to identify both similarities and differences. In general, similarities in sequence between highly divergent organisms imply functional constraint. We have used a whole-genome comparison between humans and the pufferfish, Fugu rubripes, to identify nearly 1,400 highly conserved non-coding sequences. Given the evolutionary divergence between these species, it is likely that these sequences are found in, and furthermore are essential to, all vertebrates. Most, and possibly all, of these sequences are located in and around genes that act as developmental regulators. Some of these sequences are over 90% identical across more than 500 bases, being more highly conserved than coding sequence between these two species. Despite this, we cannot find any similar sequences in invertebrate genomes. In order to begin to functionally test this set of sequences, we have used a rapid in vivo assay system using zebrafish embryos that allows tissue-specific enhancer activity to be identified. Functional data is presented for highly conserved non-coding sequences associated with four unrelated developmental regulators (SOX21, PAX6, HLXB9, and SHH), in order to demonstrate the suitability of this screen to a wide range of genes and expression patterns. Of 25 sequence elements tested around these four genes, 23 show significant enhancer activity in one or more tissues. We have identified a set of non-coding sequences that are highly conserved throughout vertebrates. They are found in clusters across the human genome, principally around genes that are implicated in the regulation of development, including many transcription factors. These highly conserved non-coding sequences are likely to form part of the genomic circuitry that uniquely defines vertebrate development.

A Fugu-Human Genome Synteny Viewer: web software for graphical display and annotation reports of synteny between Fugu genomic sequence and human genes.

A web server has been developed to access annotation and graphical reports of synteny and gene order between the Fugu genome and human genes. In this system, the assembled Fugu genomic sequences (also known as scaffolds) are annotated. The annotations for each Fugu scaffold are computed, stored and made publicly available. The annotations describe matches to human homologous genes. For each significant human gene match on the Fugu scaffold, the corresponding human chromosome map and measures of the significance of each match are given. The web-based server provides public access to these annotations and graphical displays of the results. The user is provided with a selection of views including a chromosome-colour-coded image and a table containing the details of the matches. The Fugu-Human Genome Synteny Viewer has been tested by comparing results with examples from a paper that includes a study of transcription factors, Fos and Jun encoding regions. The Fugu-human genome synteny views are available for each Fugu scaffold through the clonesearch web page located at the Fugu Genomics website (http://fugu.rfcgr.mrc.ac.uk/).

Isolation and characterisation of the corticotropin releasing factor receptor 1 (CRFR1) gene in a teleost fish, Fugu rubripes.

Corticotropin releasing factor receptor (CRF) is a member of the secretin family of the G-protein coupled receptor superfamily. These are characterised by the presence of seven transmembrane domains and six conserved cysteines that are important for receptor conformation and ligand binding. IN vertebrates two CRF receptors (CRF1 and CRF2) have been isolated and characterised. In this study the complete structure of the CRF1 receptor was isolated and partially characterised for the first time in a vertebrate using the compact genome of the Japanese pufferfish, Fugu rubripes as a model. The Fugu CRF1 receptor gene is composed of 14 exons is approximately 27 kb in length. A tissue distribution of this receptor in Fugu reveals that it is expressed mainly in liver, gonads, heart and brain, however, expression in the kidney, gut and gills was also detected. In vertebrates this receptor appears to have a different tissue distribution and its presence in the gills may indicate a new role in osmoregulatory processes.

AP1 genes in Fugu indicate a divergent transcriptional control to that of mammals.

The draft genomic sequence of the Japanese puffer fish, Fugu rubripes, has now been announced. This is the first complete sequence of a teleost fish and the second available vertebrate sequence, the first being that of human. For the first time, whole-genome comparisons between two vertebrates can be undertaken. Early analysis has suggested that there may be surprising differences in gene regulation between human and fish. In mammals, a gene commonly has several functions, and this may not always be the case in fish. Many gene families comprise more members in fish than they do in mammals, possibly because each fish gene has evolved an individual function. Complexities of gene regulation in mammals has hampered studies of all biological processes from cell proliferation to cell death. Determining the activities of the AP1 transcription factor proteins has been non-trivial. The AP1 complex typically comprises two proteins, a Jun (c-Jun, JunB, and JunD) and a Fos (c-Fos, FosB, Fra1, and Fra2). These proteins can form both homodimers and heterodimers among-themselves and can interact with additional proteins; thus, dissecting their individual roles has been difficult. We have determined that Fugu has more Jun and Fos genes than mammals, and if each proves to have a separate function, then addressing the roles of the individual AP1 proteins in Fugu may be simpler than in human.

Characterisation of a gene cluster in Fugu rubripes containing the complement component C4 gene.

In this study, we describe the characterisation of the complement component C4 gene in Fugu rubripes. The Fugu C4 gene, orthologous to the tetrapod C4 gene, encompasses a genomic span of 9702 base pairs and contains 41 exons, encoding the typical C4 three-chain polypeptide. The gene encodes a protein containing 1703 amino acids. The Fugu C4 protein demonstrates the presence of 25 conserved cysteine residues, as well as conservation of the functionally important thioester site. Complete sequencing of one cosmid and sequence scans from a cluster of 18 overlapping BAC clones, centering around the C4 gene, have identified the short-range linkage with five orthologous human genes mapping to the Major Histocompatibility Complex (MHC) including: tenascin X (TNX); cytochrome P450, subfamily XXIA, polypeptide 2 (CYP21A2); allograft inflammatory factor 1 (AIF1) and casein kinase 2, beta polypeptide (CSNK2B), all found in the MHC class III region; and retinoid X receptor, beta (RXRB),which resides in the MHC extended class II region. To date, this syntenic association of the Fugu C4 and other MHC class III region genes has not been observed in other teleost fish. Data from the recent whole-genome shotgun assemblies reveal the Fugu MHC-related cluster of genes to be flanked predominantly by genes mapping to human chromosomes 7 and 19. All of the six identified Fugu MHC-related genes have been characterised at the genomic level.

Genomic characterisation of putative growth hormone releasing hormone (GHRH) receptor genes in the teleost fish Fugu rubripes.

The genomic organisation of a putative GHRH receptor gene has been characterised in the teleost fish, Fugu rubripes. It comprises 13 exons and is at least 1.2 x larger than previously described vertebrate GHRH receptors. Sequence conservation with other vertebrate GHRH receptors is highest over the seven transmembrane domains with preservation of 8 conserved cysteines and an N-glycosylation site in the N-terminal region, which are important for receptor activation. Database mining revealed a further putative GHRH receptor in Fugu, with phylogenetic tree topology indicating that this was potentially a teleost-specific duplication event. This is the first time that the genomic organisation of a GHRH receptor gene has been characterised from a nonmammalian vertebrate.

Theatre: A software tool for detailed comparative analysis and visualization of genomic sequence.

Theatre is a web-based computing system designed for the comparative analysis of genomic sequences, especially with respect to motifs likely to be involved in the regulation of gene expression. Theatre is an interface to commonly used sequence analysis tools and biological sequence databases to determine or predict the positions of coding regions, repetitive sequences and transcription factor binding sites in families of DNA sequences. The information is displayed in a manner that can be easily understood and can reveal patterns that might not otherwise have been noticed. In addition to web-based output, Theatre can produce publication quality colour hardcopies showing predicted features in aligned genomic sequences. A case study using the p53 promoter region of four mammalian species and two fish species is described. Unlike the mammalian sequences the promoter regions in fish have not been previously predicted or characterized and we report the differences in the p53 promoter region of four mammals and that predicted for two fish species. Theatre can be accessed at http://www.hgmp.mrc.ac.uk/Registered/Webapp/theatre/.