Epigenetic mechanisms have emerged as links between prenatal environmental exposure and disease risk later in life. Here, we studied epigenetic changes associated with maternal smoking at base pair resolution by mapping DNA methylation, histone modifications, and transcription in expectant mothers and their newborn children. We found extensive global differential methylation and carefully evaluated these changes to separate environment associated from genotype-related DNA methylation changes. Differential methylation is enriched in enhancer elements and targets in particular "commuting" enhancers having multiple, regulatory interactions with distal genes. Longitudinal whole-genome bisulfite sequencing revealed that DNA methylation changes associated with maternal smoking persist over years of life. Particularly in children prenatal environmental exposure leads to chromatin transitions into a hyperactive state. Combined DNA methylation, histone modification, and gene expression analyses indicate that differential methylation in enhancer regions is more often functionally translated than methylation changes in promoters or non-regulatory elements. Finally, we show that epigenetic deregulation of a commuting enhancer targeting c-Jun N-terminal kinase 2 (JNK2) is linked to impaired lung function in early childhood.

microRNAs are an abundant class of small non-coding RNAs that control gene expression post-transcriptionally. Importantly, microRNA activity participates in the regulation of cellular processes and is a potentially valuable source of biomarkers in the diagnosis and prognosis of human diseases. Here we introduce miQPCR, an innovative method to quantify microRNAs expression by using Real-Time PCR. miQPCR exploits T4 RNA ligase activities to extend uniformly microRNAs' 3'-ends by addition of a linker-adapter. The adapter is then used as 'anchor' to prime cDNA synthesis and throughout qPCR to amplify specifically target amplicons. miQPCR is an open, adaptable and cost-effective procedure, which offers the following advantages; i) universal elongation and reverse transcription of all microRNAs; ii) Tm-adjustment of microRNA-specific primers; iii) high sensitivity and specificity in discriminating among closely related sequences and; iv) suitable for the analysis of cellular and cell-free circulating microRNAs. Analysis of cellular and cell-free circulating microRNAs secreted by rat primary hepatocytes stimulated with cytokines and growth factors identifies for the first time a widespread modulation of both microRNAs expression and secretion. Altogether, our findings suggest that the pleiotropic activity of humoral factors on microRNAs may extensively affect liver function in response to injury and regeneration.

Harmonizing quality control (QC) of large-scale second and third-generation sequencing datasets is key for enabling downstream computational and biological analyses. We present Alfred, an efficient and versatile command-line application that computes multi-sample QC metrics in a read-group aware manner, across a wide variety of sequencing assays and technologies. In addition to standard QC metrics such as GC bias, base composition, insert size and sequencing coverage distributions it supports haplotype-aware and allele-specific feature counting and feature annotation. The versatility of Alfred allows for easy pipeline integration in high-throughput settings, including DNA sequencing facilities and large-scale research initiatives, enabling continuous monitoring of sequence data quality and characteristics across samples. Alfred supports haplo-tagging of BAM/CRAM files to conduct haplotype-resolved analyses in conjunction with a variety of next-generation sequencing based assays. Alfred's companion web application enables interactive exploration of results and comparison to public datasets.

The paucity of microbiome studies at intestinal tissues has contributed to a yet limited understanding of potential viral and bacterial cofactors of colorectal cancer (CRC) carcinogenesis or progression. We analysed whole-genome sequences of CRC primary tumours, their corresponding metastases and matched normal tissue for sequences of viral, phage and bacterial species. Bacteriome analysis showed Fusobacterium nucleatum, Streptococcus sanguinis, F. Hwasookii, Anaerococcus mediterraneensis and further species enriched in primary CRCs. The primary CRC of one patient was enriched for F. alocis, S. anginosus, Parvimonas micra and Gemella sp. 948. Enrichment of Escherichia coli strains IAI1, SE11, K-12 and M8 was observed in metastases together with coliphages enterobacteria phage φ80 and Escherichia phage VT2φ_272. Virome analysis showed that phages were the most preponderant viral species (46%), the main families being Myoviridae, Siphoviridae and Podoviridae. Primary CRCs were enriched for bacteriophages, showing five phages (Enterobacteria, Bacillus, Proteus, Streptococcus phages) together with their pathogenic hosts in contrast to normal tissues. The most frequently detected, and Blast-confirmed, viruses included human endogenous retrovirus K113, human herpesviruses 7 and 6B, Megavirus chilensis, cytomegalovirus (CMV) and Epstein-Barr virus (EBV), with one patient showing EBV enrichment in primary tumour and metastases. EBV was PCR-validated in 80 pairs of CRC primary tumour and their corresponding normal tissues; in 21 of these pairs (26.3%), it was detectable in primary tumours only. The number of viral species was increased and bacterial species decreased in CRCs compared with normal tissues, and we could discriminate primary CRCs from metastases and normal tissues by applying the Hutcheson t-test on the Shannon indices based on viral and bacterial species. Taken together, our results descriptively support hypotheses on microorganisms as potential (co)risk factors of CRC and extend putative suggestions on critical microbiome species in CRC metastasis.

Neuroblastoma is the most common extra-cranial solid tumor of early childhood. Standard therapies are not effective in case of poor prognosis and chemotherapy resistance. To improve drug therapy, it is imperative to discover new targets that play a substantial role in tumorigenesis of neuroblastoma. The mitotic machinery is an attractive target for therapeutic interventions and inhibitors can be developed to target mitotic entry, spindle apparatus, spindle activation checkpoint, and mitotic exit. We present an elaborate analysis pipeline to determine cancer specific therapeutic targets by first performing a focused gene expression analysis to select genes followed by a gene knockdown screening assay of live cells. We interrogated gene expression studies of neuroblastoma tumors and selected 240 genes relevant for tumorigenesis and cell cycle. With these genes we performed time-lapse screening of gene knockdowns in neuroblastoma cells. We classified cellular phenotypes and used the temporal context of the perturbation effect to determine the sequence of events, particularly the mitotic entry preceding cell death. Based upon this phenotype kinetics from the gene knockdown screening, we inferred dynamic gene functions in mitosis and cell proliferation. We identified six genes (DLGAP5, DSCC1, SMO, SNRPD1, SSBP1, and UBE2C) with a vital role in mitosis and these are promising therapeutic targets for neuroblastoma. Images and movies of every time point of all screened genes are available at https://ichip.bioquant.uni-heidelberg.de.

Follicular dendritic cell (FDC)-sarcoma is a rare neoplasm with morphologic and phenotypic features of FDCs. It shows an extremely heterogeneous morphology, therefore, its diagnosis relys on the phenotype of tumor cells. Aim of the present study was the identification of new specific markers for FDC-sarcoma by whole transcriptome sequencing (WTS). Candidate markers were selected based on gene expression level and biological function. Immunohistochemistry was performed on reactive tonsils, on 22 cases of FDC-sarcomas and 214 control cases including 114 carcinomas, 87 soft tissue tumors, 5 melanomas, 5 thymomas and 3 interdigitating dendritic cell sarcomas. FDC secreted protein (FDCSP) and Serglycin (SRGN) proved to be specific markers of FDC and related tumor. They showed better specificity and sensitivity values than some well known markers used in FDC sarcoma diagnosis (specificity: 98.6%, and 100%, respectively; sensitivity: 72.73% and 68.18%, respectively). In our cohorts CXCL13, CD21, CD35, FDCSP and SRGN were the best markers for FDC-sarcoma diagnosis and could discriminate 21/22 FDC sarcomas from other mesenchymal tumors by linear discriminant analysis. In summary, by WTS we identified two novel FDC markers and by the analysis of a wide cohort of cases and controls we propose an efficient marker panel for the diagnosis of this rare and enigmatic tumor.

The transcriptome needs to be tightly regulated by mechanisms that include transcription factors, enhancers, and repressors as well as non-coding RNAs. Besides this dynamic regulation, a large part of phenotypic variability of eukaryotes is expressed through changes in gene transcription caused by genetic variation. In this study, we evaluate genome-wide structural genomic variants (SVs) and their association with gene expression in the human heart. We detected 3,898 individual SVs affecting all classes of gene transcripts (e.g., mRNA, miRNA, lncRNA) and regulatory genomic regions (e.g., enhancer or TFBS). In a cohort of patients (n = 50) with dilated cardiomyopathy (DCM), 80,635 non-protein-coding elements of the genome are deleted or duplicated by SVs, containing 3,758 long non-coding RNAs and 1,756 protein-coding transcripts. 65.3% of the SV-eQTLs do not harbor a significant SNV-eQTL, and for the regions with both classes of association, we find similar effect sizes. In case of deleted protein-coding exons, we find downregulation of the associated transcripts, duplication events, however, do not show significant changes over all events. In summary, we are first to describe the genomic variability associated with SVs in heart failure due to DCM and dissect their impact on the transcriptome. Overall, SVs explain up to 7.5% of the variation of cardiac gene expression, underlining the importance to study human myocardial gene expression in the context of the individual genome. This has immediate implications for studies on basic mechanisms of cardiac maladaptation, biomarkers, and (gene) therapeutic studies alike.

The Grainyhead-like (GRHL) transcription factors have been linked to many different types of cancer. However, no previous study has attempted to investigate potential correlations in expression of different GRHL genes in this context. Furthermore, there is very little information concerning damaging mutations and/or single nucleotide polymorphisms in GRHL genes that may be linked to cancer.

Although smallpox has been known for centuries, the oldest available variola virus strains were isolated in the early 1940s. At that time, large regions of the world were already smallpox-free. Therefore, genetic information of these strains can represent only the very last fraction of a long evolutionary process. Based on the genomes of 48 strains, two clades are differentiated: Clade 1 includes variants of variola major, and clade 2 includes West African and variola minor (Alastrim) strains. Recently, the genome of an almost 400-year-old Lithuanian mummy was determined, which fell basal to all currently sequenced strains of variola virus on phylogenetic trees. Here, we determined two complete variola virus genomes from human tissues kept in a museum in Prague dating back 60 and 160 years, respectively. Moreover, mass spectrometry-based proteomic, chemical, and microscopic examinations were performed. The 60-year-old specimen was most likely an importation from India, a country with endemic smallpox at that time. The genome of the 160-year-old specimen is related to clade 2 West African and variola minor strains. This sequence likely represents a new endemic European variant of variola virus circulating in the midst of the 19th century in Europe.

The mechanisms underlying T-ALL relapse remain essentially unknown. Multilevel-omics in 38 matched pairs of initial and relapsed T-ALL revealed 18 (47%) type-1 (defined by being derived from the major ancestral clone) and 20 (53%) type-2 relapses (derived from a minor ancestral clone). In both types of relapse, we observed known and novel drivers of multidrug resistance including MDR1 and MVP, NT5C2 and JAK-STAT activators. Patients with type-1 relapses were specifically characterized by IL7R upregulation. In remarkable contrast, type-2 relapses demonstrated (1) enrichment of constitutional cancer predisposition gene mutations, (2) divergent genetic and epigenetic remodeling, and (3) enrichment of somatic hypermutator phenotypes, related to BLM, BUB1B/PMS2 and TP53 mutations. T-ALLs that later progressed to type-2 relapses exhibited a complex subclonal architecture, unexpectedly, already at the time of initial diagnosis. Deconvolution analysis of ATAC-Seq profiles showed that T-ALLs later developing into type-1 relapses resembled a predominant immature thymic T-cell population, whereas T-ALLs developing into type-2 relapses resembled a mixture of normal T-cell precursors. In sum, our analyses revealed fundamentally different mechanisms driving either type-1 or type-2 T-ALL relapse and indicate that differential capacities of disease evolution are already inherent to the molecular setup of the initial leukemia.

We compared 24 primary pediatric T-cell acute lymphoblastic leukemias (T-ALL) collected at the time of initial diagnosis and relapse from 12 patients and 24 matched patient-derived xenografts (PDXs). DNA methylation profile was preserved in PDX mice in 97.5% of the promoters (ρ = 0.99). Similarly, the genome-wide chromatin accessibility (ATAC-Seq) was preserved remarkably well (ρ = 0.96). Interestingly, both the ATAC regions, which showed a significant decrease in accessibility in PDXs and the regions hypermethylated in PDXs, were associated with immune response, which might reflect the immune deficiency of the mice and potentially the incomplete interaction between murine cytokines and human receptors. The longitudinal approach of this study allowed an observation that samples collected from patients who developed a type 1 relapse (clonal mutations maintained at relapse) preserved their genomic composition; whereas in patients who developed a type 2 relapse (subset of clonal mutations lost at relapse), the preservation of the leukemia's composition was more variable. In sum, this study underlines the remarkable genomic stability, and for the first time documents the preservation of the epigenomic landscape in T-ALL-derived PDX models.

Whole-genome and whole-exome sequencing of individual patients allow the study of rare and potentially causative genetic variation. In this study, we sequenced DNA of a trio comprising a boy with very-early-onset inflammatory bowel disease (veoIBD) and his unaffected parents. We identified a rare, X-linked missense variant in the NAPDH oxidase NOX1 gene (c.C721T, p.R241C) in heterozygous state in the mother and in hemizygous state in the patient. We discovered that, in addition, the patient was homozygous for a common missense variant in the CYBA gene (c.T214C, p.Y72H). CYBA encodes the p22phox protein, a cofactor for NOX1. Functional assays revealed reduced cellular ROS generation and antibacterial capacity of NOX1 and p22phox variants in intestinal epithelial cells. Moreover, the identified NADPH oxidase complex variants affected NOD2-mediated immune responses, and p22phox was identified as a novel NOD2 interactor. In conclusion, we detected missense variants in a veoIBD patient that disrupt the host response to bacterial challenges and reduce protective innate immune signaling via NOD2. We assume that the patient's individual genetic makeup favored disturbed intestinal mucosal barrier function.

Medulloblastoma is associated with rare hereditary cancer predisposition syndromes; however, consensus medulloblastoma predisposition genes have not been defined and screening guidelines for genetic counselling and testing for paediatric patients are not available. We aimed to assess and define these genes to provide evidence for future screening guidelines.

Genomic structural variants (SVs) are abundant in humans, differing from other forms of variation in extent, origin and functional impact. Despite progress in SV characterization, the nucleotide resolution architecture of most SVs remains unknown. We constructed a map of unbalanced SVs (that is, copy number variants) based on whole genome DNA sequencing data from 185 human genomes, integrating evidence from complementary SV discovery approaches with extensive experimental validations. Our map encompassed 22,025 deletions and 6,000 additional SVs, including insertions and tandem duplications. Most SVs (53%) were mapped to nucleotide resolution, which facilitated analysing their origin and functional impact. We examined numerous whole and partial gene deletions with a genotyping approach and observed a depletion of gene disruptions amongst high frequency deletions. Furthermore, we observed differences in the size spectra of SVs originating from distinct formation mechanisms, and constructed a map of SV hotspots formed by common mechanisms. Our analytical framework and SV map serves as a resource for sequencing-based association studies.

T-cell/histiocyte-rich large B-cell lymphoma is a rare aggressive lymphoma showing histopathological overlap with nodular lymphocyte-predominant Hodgkin lymphoma. Despite differences in tumor microenvironment and clinical behavior, the tumor cells of both entities show remarkable similarities, suggesting that both lymphomas might represent a spectrum of the same disease. To address this issue, we investigated whether these entities share mutations. Ultra-deep targeted resequencing of six typical and 11 histopathological variants of nodular lymphocyte-predominant Hodgkin lymphoma, and nine cases of T-cell/histiocyte-rich large B-cell lymphoma revealed that genes recurrently mutated in nodular lymphocyte-predominant Hodgkin lymphoma are affected by mutations at similar frequencies in T-cell/histiocyte-rich large B-cell lymphoma. The most recurrently mutated genes were JUNB, DUSP2, SGK1, SOCS1 and CREBBP, which harbored mutations more frequently in T-cell/histiocyte-rich large B-cell lymphoma and the histopathological variants of nodular lymphocyte-predominant Hodgkin lymphoma than in its typical form. Mutations in JUNB, DUSP2, SGK1 and SOCS1 were highly enriched for somatic hypermutation hotspot sites, suggesting an important role of aberrant somatic hypermutation in the generation of these somatic mutations and thus in the pathogenesis of both lymphoma entities. Mutations in JUNB are generally rarely observed in malignant lymphomas and thus are relatively specific for nodular lymphocyte-predominant Hodgkin lymphoma and T-cell/histiocyte-rich large B-cell lymphoma at such high frequencies (5/17 and 5/9 cases with JUNB mutations, respectively). Taken together, the findings of the present study further support a close relationship between T-cell/histiocyte-rich large B-cell lymphoma and nodular lymphocyte-predominant Hodgkin lymphoma by showing that they share highly recurrent genetic lesions.

HeLa is the most widely used model cell line for studying human cellular and molecular biology. To date, no genomic reference for this cell line has been released, and experiments have relied on the human reference genome. Effective design and interpretation of molecular genetic studies performed using HeLa cells require accurate genomic information. Here we present a detailed genomic and transcriptomic characterization of a HeLa cell line. We performed DNA and RNA sequencing of a HeLa Kyoto cell line and analyzed its mutational portfolio and gene expression profile. Segmentation of the genome according to copy number revealed a remarkably high level of aneuploidy and numerous large structural variants at unprecedented resolution. Some of the extensive genomic rearrangements are indicative of catastrophic chromosome shattering, known as chromothripsis. Our analysis of the HeLa gene expression profile revealed that several pathways, including cell cycle and DNA repair, exhibit significantly different expression patterns from those in normal human tissues. Our results provide the first detailed account of genomic variants in the HeLa genome, yielding insight into their impact on gene expression and cellular function as well as their origins. This study underscores the importance of accounting for the strikingly aberrant characteristics of HeLa cells when designing and interpreting experiments, and has implications for the use of HeLa as a model of human biology.

Spondyloepimetaphyseal dysplasia with joint laxity-leptodactylic type (SEMDJL2) is an autosomal dominant skeletal dysplasia which is characterized by midface hypoplasia, short stature, joint laxity with dislocations, genua valga, progressive scoliosis, and slender fingers. Recently, heterozygous missense mutations in KIF22, a gene which encodes a member of the kinesin-like protein family, have been identified in sporadic as well as familial cases of SEMDJL2. In the present study homozygosity mapping and whole-exome sequencing were combined to analyze a consanguineous family with a phenotype resembling SEMDJL2. We identified homozygous missense mutations in the two nearby genes NIN (Ninein) and POLE2 (DNA polymerase epsilon subunit B) which segregate with the disease in the family and were not present in 500 healthy control individuals and in the 1094 control individuals contained within the 1000-genomes database. We present several lines of evidence that mutant Ninein is most likely causative for the SEMDJL2-like phenotype. The centrosomal protein NIN shows a functional relationship with KIF22 and other proteins associated with chromosome congression/movement, centrosomal function, and ciliogenesis, which have been associated with skeletal dysplasias. Moreover, compound heterozygous missense mutations at more N-terminal positions of Ninein have very recently been identified in a family with microcephalic primordial dwarfism. Together with the present report this strongly supports a fundamental role of Ninein in skeletal development.

Genomic structural variation (SV) is a major determinant for phenotypic variation. Although it has been extensively studied in humans, the nucleotide resolution structure of SVs within the widely used model organism Drosophila remains unknown. We report a highly accurate, densely validated map of unbalanced SVs comprising 8962 deletions and 916 tandem duplications in 39 lines derived from short-read DNA sequencing in a natural population (the "Drosophila melanogaster Genetic Reference Panel," DGRP). Most SVs (>90%) were inferred at nucleotide resolution, and a large fraction was genotyped across all samples. Comprehensive analyses of SV formation mechanisms using the short-read data revealed an abundance of SVs formed by mobile element and nonhomologous end-joining-mediated rearrangements, and clustering of variants into SV hotspots. We further observed a strong depletion of SVs overlapping genes, which, along with population genetics analyses, suggests that these SVs are often deleterious. We inferred several gene fusion events also highlighting the potential role of SVs in the generation of novel protein products. Expression quantitative trait locus (eQTL) mapping revealed the functional impact of our high-resolution SV map, with quantifiable effects at >100 genic loci. Our map represents a resource for population-level studies of SVs in an important model organism.

Genomic rearrangements are thought to occur progressively during tumor development. Recent findings, however, suggest an alternative mechanism, involving massive chromosome rearrangements in a one-step catastrophic event termed chromothripsis. We report the whole-genome sequencing-based analysis of a Sonic-Hedgehog medulloblastoma (SHH-MB) brain tumor from a patient with a germline TP53 mutation (Li-Fraumeni syndrome), uncovering massive, complex chromosome rearrangements. Integrating TP53 status with microarray and deep sequencing-based DNA rearrangement data in additional patients reveals a striking association between TP53 mutation and chromothripsis in SHH-MBs. Analysis of additional tumor entities substantiates a link between TP53 mutation and chromothripsis, and indicates a context-specific role for p53 in catastrophic DNA rearrangements. Among these, we observed a strong association between somatic TP53 mutations and chromothripsis in acute myeloid leukemia. These findings connect p53 status and chromothripsis in specific tumor types, providing a genetic basis for understanding particularly aggressive subtypes of cancer.

The incomplete identification of structural variants (SVs) from whole-genome sequencing data limits studies of human genetic diversity and disease association. Here, we apply a suite of long-read, short-read, strand-specific sequencing technologies, optical mapping, and variant discovery algorithms to comprehensively analyze three trios to define the full spectrum of human genetic variation in a haplotype-resolved manner. We identify 818,054 indel variants (<50 bp) and 27,622 SVs (≥50 bp) per genome. We also discover 156 inversions per genome and 58 of the inversions intersect with the critical regions of recurrent microdeletion and microduplication syndromes. Taken together, our SV callsets represent a three to sevenfold increase in SV detection compared to most standard high-throughput sequencing studies, including those from the 1000 Genomes Project. The methods and the dataset presented serve as a gold standard for the scientific community allowing us to make recommendations for maximizing structural variation sensitivity for future genome sequencing studies.