In excess of 12% of human cancer incidents have a viral cofactor. Epidemiological studies of idiopathic human cancers indicate that additional tumor viruses remain to be discovered. Recent advances in sequencing technology have enabled systematic screenings of human tumor transcriptomes for viral transcripts. However, technical problems such as low abundances of viral transcripts in large volumes of sequencing data, viral sequence divergence, and homology between viral and human factors significantly confound identification of tumor viruses. We have developed a novel computational approach for detecting viral transcripts in human cancers that takes the aforementioned confounding factors into account and is applicable to a wide variety of viruses and tumors. We apply the approach to conducting the first systematic search for viruses in neuroblastoma, the most common cancer in infancy. The diverse clinical progression of this disease as well as related epidemiological and virological findings are highly suggestive of a pathogenic cofactor. However, a viral etiology of neuroblastoma is currently contested. We mapped 14 transcriptomes of neuroblastoma as well as positive and negative controls to the human and all known viral genomes in order to detect both known and unknown viruses. Analysis of controls, comparisons with related methods, and statistical estimates demonstrate the high sensitivity of our approach. Detailed investigation of putative viral transcripts within neuroblastoma samples did not provide evidence for the existence of any known human viruses. Likewise, de-novo assembly and analysis of chimeric transcripts did not result in expression signatures associated with novel human pathogens. While confounding factors such as sample dilution or viral clearance in progressed tumors may mask viral cofactors in the data, in principle, this is rendered less likely by the high sensitivity of our approach and the number of biological replicates analyzed. Therefore, our results suggest that frequent viral cofactors of metastatic neuroblastoma are unlikely.

Cyanobacteria constitute a serious threat to freshwater ecosystems by producing toxic secondary metabolites, e.g. microcystins. These microcystins have been shown to harm livestock, pets and humans and to affect ecosystem service and functioning. Cyanobacterial blooms are increasing worldwide in intensity and frequency due to eutrophication and global warming. However, Daphnia, the main grazer of planktonic algae and cyanobacteria, has been shown to be able to suppress bloom-forming cyanobacteria and to adapt to cyanobacteria that produce microcystins. Since Daphnia's genome was published only recently, it is now possible to elucidate the underlying molecular mechanisms of microcystin tolerance of Daphnia.

The diagnoses of retinitis pigmentosa (RP) and stationary night blindness (CSNB) are two distinct clinical entities belonging to a group of clinically and genetically heterogeneous retinal diseases. The current study focused on the identification of causative mutations in the RP-affected index patient and in several members of the same family that reported a phenotype resembling CSNB. Ophthalmological examinations of the index patient confirmed a typical form of RP. In contrast, clinical characterizations and ERGs of another affected family member showed the Riggs-type CSNB lacking signs of RP. Applying whole exome sequencing we detected the non-synonymous substitution c.337G > A, p.E113 K in the rhodopsin (RHO) gene. The mutation co-segregated with the diseases. The identification of the pathogenic variant p.E113 K is the first description of a naturally-occurring mutation in the Schiff base counterion of RHO in human patients. The heterozygous mutation c.337G > A in exon 1 was confirmed in the index patient as well as in five CSNB-affected relatives. This pathogenic sequence change was excluded in a healthy family member and in 199 ethnically matched controls. Our findings suggest that a mutation in the biochemically well-characterized counterion p.E113 in RHO can be associated with RP or Riggs-type CSNB, even within the same family.

In guanosine diphosphate (GDP)-mannose pyrophosphorylase A (GMPPA), we identified a homozygous nonsense mutation that segregated with achalasia and alacrima, delayed developmental milestones, and gait abnormalities in a consanguineous Pakistani pedigree. Mutations in GMPPA were subsequently found in ten additional individuals from eight independent families affected by the combination of achalasia, alacrima, and neurological deficits. This autosomal-recessive disorder shows many similarities with triple A syndrome, which is characterized by achalasia, alacrima, and variable neurological deficits in combination with adrenal insufficiency. GMPPA is a largely uncharacterized homolog of GMPPB. GMPPB catalyzes the formation of GDP-mannose, which is an essential precursor of glycan moieties of glycoproteins and glycolipids and is associated with congenital and limb-girdle muscular dystrophies with hypoglycosylation of α-dystroglycan. Surprisingly, GDP-mannose pyrophosphorylase activity was unchanged and GDP-mannose levels were strongly increased in lymphoblasts of individuals with GMPPA mutations. This suggests that GMPPA might serve as a GMPPB regulatory subunit mediating feedback inhibition of GMPPB instead of displaying catalytic enzyme activity itself. Thus, a triple-A-like syndrome can be added to the growing list of congenital disorders of glycosylation, in which dysregulation rather than mere enzyme deficiency is the basal pathophysiological mechanism.

A complex network of genes determines sex in mammals. Here, we studied a European roe deer with an intersex phenotype that was consistent with a XY genotype with incomplete male-determination. Whole genome sequencing and quantitative real-time PCR analyses revealed a triple dose of the SOX9 gene, allowing insights into a new genetic defect in a wild animal.

The specific functions of sensory systems depend on the tissue-specific expression of genes that code for molecular sensor proteins that are necessary for stimulus detection and membrane signaling. Using the Next Generation Sequencing technique (RNA-Seq), we analyzed the complete transcriptome of the trigeminal ganglia (TG) and dorsal root ganglia (DRG) of adult mice. Focusing on genes with an expression level higher than 1 FPKM (fragments per kilobase of transcript per million mapped reads), we detected the expression of 12984 genes in the TG and 13195 in the DRG. To analyze the specific gene expression patterns of the peripheral neuronal tissues, we compared their gene expression profiles with that of the liver, brain, olfactory epithelium, and skeletal muscle. The transcriptome data of the TG and DRG were scanned for virtually all known G-protein-coupled receptors (GPCRs) as well as for ion channels. The expression profile was ranked with regard to the level and specificity for the TG. In total, we detected 106 non-olfactory GPCRs and 33 ion channels that had not been previously described as expressed in the TG. To validate the RNA-Seq data, in situ hybridization experiments were performed for several of the newly detected transcripts. To identify differences in expression profiles between the sensory ganglia, the RNA-Seq data of the TG and DRG were compared. Among the differentially expressed genes (> 1 FPKM), 65 and 117 were expressed at least 10-fold higher in the TG and DRG, respectively. Our transcriptome analysis allows a comprehensive overview of all ion channels and G protein-coupled receptors that are expressed in trigeminal ganglia and provides additional approaches for the investigation of trigeminal sensing as well as for the physiological and pathophysiological mechanisms of pain.

Colletotrichum species are fungal pathogens that devastate crop plants worldwide. Host infection involves the differentiation of specialized cell types that are associated with penetration, growth inside living host cells (biotrophy) and tissue destruction (necrotrophy). We report here genome and transcriptome analyses of Colletotrichum higginsianum infecting Arabidopsis thaliana and Colletotrichum graminicola infecting maize. Comparative genomics showed that both fungi have large sets of pathogenicity-related genes, but families of genes encoding secreted effectors, pectin-degrading enzymes, secondary metabolism enzymes, transporters and peptidases are expanded in C. higginsianum. Genome-wide expression profiling revealed that these genes are transcribed in successive waves that are linked to pathogenic transitions: effectors and secondary metabolism enzymes are induced before penetration and during biotrophy, whereas most hydrolases and transporters are upregulated later, at the switch to necrotrophy. Our findings show that preinvasion perception of plant-derived signals substantially reprograms fungal gene expression and indicate previously unknown functions for particular fungal cell types.

Autosomal-recessive primary microcephaly (MCPH) is a rare congenital disorder characterized by intellectual disability, reduced brain and head size, but usually without defects in cerebral cortical architecture, and other syndromic abnormalities. MCPH is heterogeneous. The underlying genes of the seven known loci code for centrosomal proteins. We studied a family from northern Pakistan with two microcephalic children using homozygosity mapping and found suggestive linkage for regions on chromosomes 2, 4, and 9. We sequenced two positional candidate genes and identified a homozygous frameshift mutation in the gene encoding the 135 kDa centrosomal protein (CEP135), located in the linkage interval on chromosome 4, in both affected children. Post hoc whole-exome sequencing corroborated this mutation's identification as the causal variant. Fibroblasts obtained from one of the patients showed multiple and fragmented centrosomes, disorganized microtubules, and reduced growth rate. Similar effects were reported after knockdown of CEP135 through RNA interference; we could provoke them also by ectopic overexpression of the mutant protein. Our findings suggest an additional locus for MCPH at HSA 4q12 (MCPH8), further strengthen the role of centrosomes in the development of MCPH, and place CEP135 among the essential components of this important organelle in particular for a normal neurogenesis.

Evolutionary dynamics of structural genetic variation in lineages of hybrid origin is not well explored, although structural mutations may increase in controlled hybrid crosses. We therefore tested whether structural variants accumulate in a fish of recent hybrid origin, invasive Cottus, relative to both parental species Cottus rhenanus and Cottus perifretum. Copy-number variation in exons of 10,979 genes was assessed using comparative genome hybridization arrays. Twelve genes showed significantly higher copy numbers in invasive Cottus compared to both parents. This coincided with increased expression for three genes related to vision, detoxification and muscle development, suggesting possible gene dosage effects. Copy number increases of putative transposons were assessed by comparative mapping of genomic DNA reads against a de novo assembly of 1,005 repetitive elements. In contrast to exons, copy number increases of repetitive elements were common (20.7%) in invasive Cottus, whereas decrease was very rare (0.01%). Among the increased repetitive elements, 53.8% occurred at higher numbers in C. perifretum compared to C. rhenanus, while only 1.4% were more abundant in C. rhenanus. This implies a biased mutational process that amplifies genetic material from one ancestor. To assess the frequency of de novo mutations through hybridization, we screened 64 laboratory-bred F2 offspring between the parental species for copy-number changes at five candidate loci. We found no evidence for new structural variants, indicating that they are too rare to be detected given our sampling scheme. Instead, they must have accumulated over more generations than we observed in a controlled cross.

Bloom syndrome, caused by biallelic mutations in BLM, is characterized by prenatal-onset growth deficiency, short stature, an erythematous photosensitive malar rash, and increased cancer predisposition. Diagnostically, a hallmark feature is the presence of increased sister chromatid exchanges (SCEs) on cytogenetic testing. Here, we describe biallelic mutations in TOP3A in ten individuals with prenatal-onset growth restriction and microcephaly. TOP3A encodes topoisomerase III alpha (TopIIIα), which binds to BLM as part of the BTRR complex, and promotes dissolution of double Holliday junctions arising during homologous recombination. We also identify a homozygous truncating variant in RMI1, which encodes another component of the BTRR complex, in two individuals with microcephalic dwarfism. The TOP3A mutations substantially reduce cellular levels of TopIIIα, and consequently subjects' cells demonstrate elevated rates of SCE. Unresolved DNA recombination and/or replication intermediates persist into mitosis, leading to chromosome segregation defects and genome instability that most likely explain the growth restriction seen in these subjects and in Bloom syndrome. Clinical features of mitochondrial dysfunction are evident in several individuals with biallelic TOP3A mutations, consistent with the recently reported additional function of TopIIIα in mitochondrial DNA decatenation. In summary, our findings establish TOP3A mutations as an additional cause of prenatal-onset short stature with increased cytogenetic SCEs and implicate the decatenation activity of the BTRR complex in their pathogenesis.

Nesprin-2 is a nuclear envelope component and provides a link between cytoskeletal components of the cytoplasm and the nucleoplasm. Several isoforms are generated from its gene Syne2. Loss of the largest isoform Nesprin-2 Giant in mice is associated with a skin phenotype and altered wound healing, loss of C-terminal isoforms in mice leads to cardiomyopathies and neurological defects. Here we attempted to establish mice with an inducible knockout of all Nesprin-2 isoforms by inserting shRNA encoding sequences targeting the N- and C-terminus into the ROSA26 locus of mice. This caused early embryonic death of the animals harboring the mutant allele, which was presumably due to leaky expression of the shRNAs. Mutant embryos were only observed before E13. They had an altered appearance and were smaller in size than their wild type littermates. From this we conclude that the Nesprin-2 gene function is crucial during embryonic growth, differentiation and organogenesis.

Rare diseases are often misdiagnosed or receive a delayed diagnosis; thus, unfortunately, affected individuals may not receive optimal medical management. Here, we report a case of two siblings with a severe phenotype of progressive pseudorheumatoid dysplasia (PPD). Their onset of symptoms began at the age of 3 yr. Both were neglected in the past, and the patients presented with a very severe phenotype and unmitigated natural history. PPD is a rare autosomal recessive skeletal dysplasia characterized by progressive joint stiffness, swelling, and pain. Because of observed muscle wasting, weakness, and the lack of laboratory testing, the case had been initially misdiagnosed by the local physicians. We aimed to provide diagnostic support by a targeted next-generation sequencing gene panel (Illumina TruSight One) for Mendelian diseases (Mendeliome), and we identified a homozygous frameshift mutation in the gene WISP3 (c.868_869delAG, p.Ser290Leufs*12). Thus, early diagnosis and intervention may have decreased the severity and complication of the disease.

Very recently, compound heterozygous loss-of-function mutations in TELO2 were shown to underlie the newly-described You-Hoover-Fong syndrome. TELO2 forms part of the co-chaperone triple T complex (TTT complex), which plays an important role in the maturation and stabilization of the phosphatidylinositol 3-kinase-related protein kinases (PIKKs). Patients with mutations in TELO2 present with microcephaly and associated intellectual disability, postnatal growth retardation and dysmorphic features. Here, we describe Danish sisters with two novel mutations in TELO2. In particular, we highlight the clinical features of the 22-year index patient, which are more severe than the original patients described, thereby expanding the clinical spectrum of YHFS.

Dysregulated miRNA expression and mutation of genes involved in miRNA biogenesis have been reported in motor neuron diseases including spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Therefore, identifying molecular mechanisms governing miRNA expression is important to understand these diseases. Here, we report that expression of DROSHA, which is a critical enzyme in the microprocessor complex and essential for miRNA biogenesis, is reduced in motor neurons from an SMA mouse model. We show that DROSHA is degraded by neuronal activity induced autophagy machinery, which is also dysregulated in SMA. Blocking neuronal activity or the autophagy-lysosome pathway restores DROSHA levels in SMA motor neurons. Moreover, reducing DROSHA levels enhances axonal growth. As impaired axonal growth is a well described phenotype of SMA motor neurons, these data suggest that DROSHA reduction by autophagy may mitigate the phenotype of SMA. In summary, these findings suggest that autophagy regulates RNA metabolism and neuronal growth via the DROSHA/miRNA pathway and this pathway is dysregulated in SMA.

Barley (Hordeum vulgare) is an established model to study domestication of the Fertile Crescent cereals. Recent molecular data suggested that domesticated barley genomes consist of the ancestral blocks descending from multiple wild barley populations. However, the relationship between the mosaic ancestry patterns and the process of domestication itself remained unclear. To address this knowledge gap, we identified candidate domestication genes using selection scans based on targeted resequencing of 433 wild and domesticated barley accessions. We conducted phylogenetic, population structure, and ancestry analyses to investigate the origin of the domesticated barley haplotypes separately at the neutral and candidate domestication loci. We discovered multiple selective sweeps that occurred on all barley chromosomes during domestication in the background of several ancestral wild populations. The ancestry analyses demonstrated that, although the ancestral blocks of the domesticated barley genomes were descended from all over the Fertile Crescent, the candidate domestication loci originated specifically in its eastern and western parts. These findings provided the first molecular evidence implicating multiple wild or protodomesticated lineages in the process of barley domestication initiated in the Levantine and Zagros clusters of the origin of agriculture.

Mismatch repair (MMR)-deficiency increases the risk of colorectal tumorigenesis. To determine whether the tumors develop on a normal or disturbed epigenetic background and how radiation affects this, we quantified genome-wide histone H3 methylation profiles in macroscopic normal intestinal tissue of young radiated and untreated MMR-deficient VCMsh2LoxP/LoxP (Msh2-/-) mice months before tumor onset.

Congenital lower urinary-tract obstruction (LUTO) is caused by anatomical blockage of the bladder outflow tract or by functional impairment of urinary voiding. About three out of 10,000 pregnancies are affected. Although several monogenic causes of functional obstruction have been defined, it is unknown whether congenital LUTO caused by anatomical blockage has a monogenic cause. Exome sequencing in a family with four affected individuals with anatomical blockage of the urethra identified a rare nonsense variant (c.2557C>T [p.Arg853∗]) in BNC2, encoding basonuclin 2, tracking with LUTO over three generations. Re-sequencing BNC2 in 697 individuals with LUTO revealed three further independent missense variants in three unrelated families. In human and mouse embryogenesis, basonuclin 2 was detected in lower urinary-tract rudiments. In zebrafish embryos, bnc2 was expressed in the pronephric duct and cloaca, analogs of the mammalian lower urinary tract. Experimental knockdown of Bnc2 in zebrafish caused pronephric-outlet obstruction and cloacal dilatation, phenocopying human congenital LUTO. Collectively, these results support the conclusion that variants in BNC2 are strongly implicated in LUTO etiology as a result of anatomical blockage.

Transcription factor NRF2, encoded by NFE2L2, is the master regulator of defense against stress in mammalian cells. Somatic mutations of NFE2L2 leading to NRF2 accumulation promote cell survival and drug resistance in cancer cells. Here we show that the same mutations as inborn de novo mutations cause an early onset multisystem disorder with failure to thrive, immunodeficiency and neurological symptoms. NRF2 accumulation leads to widespread misregulation of gene expression and an imbalance in cytosolic redox balance. The unique combination of white matter lesions, hypohomocysteinaemia and increased G-6-P-dehydrogenase activity will facilitate early diagnosis and therapeutic intervention of this novel disorder.The NRF2 transcription factor regulates the response to stress in mammalian cells. Here, the authors show that activating mutations in NRF2, commonly found in cancer cells, are found in four patients with a multisystem disorder characterized by immunodeficiency and neurological symptoms.

Processes like cellular senescence are characterized by complex events giving rise to heterogeneous cell populations. However, the early molecular events driving this cascade remain elusive. We hypothesized that senescence entry is triggered by an early disruption of the cells' three-dimensional (3D) genome organization. To test this, we combined Hi-C, single-cell and population transcriptomics, imaging, and in silico modeling of three distinct cells types entering senescence. Genes involved in DNA conformation maintenance are suppressed upon senescence entry across all cell types. We show that nuclear depletion of the abundant HMGB2 protein occurs early on the path to senescence and coincides with the dramatic spatial clustering of CTCF. Knocking down HMGB2 suffices for senescence-induced CTCF clustering and for loop reshuffling, while ectopically expressing HMGB2 rescues these effects. Our data suggest that HMGB2-mediated genomic reorganization constitutes a primer for the ensuing senescent program.

Deciphering the evolution of cancer cells under therapeutic pressure is a crucial step to understand the mechanisms that lead to treatment resistance. To this end, we analyzed whole-exome sequencing data of eight chronic lymphocytic leukemia (CLL) patients that developed resistance upon BCL2-inhibition by venetoclax. Here, we report recurrent mutations in BTG1 (2 patients) and homozygous deletions affecting CDKN2A/B (3 patients) that developed during treatment, as well as a mutation in BRAF and a high-level focal amplification of CD274 (PD-L1) that might pinpoint molecular aberrations offering structures for further therapeutic interventions.