Evolution and implications of de novo genes in humans

Genes and translated open reading frames (ORFs) that emerged de novo from previously non-coding sequences provide species with opportunities for adaptation. When aberrantly activated, some human-specific de novo genes and ORFs have disease-promoting properties—for instance, driving tumour growth. Thousands of putative de novo coding sequences have been described in humans, but we still do not know what fraction of those ORFs has readily acquired a function. Here, we discuss the challenges and controversies surrounding the detection, mechanisms of origin, annotation, validation and characterization of de novo genes and ORFs. Through manual curation of literature and databases, we provide a thorough table with most de novo genes reported for humans to date. We re-evaluate each locus by tracing the enabling mutations and list proposed disease associations, protein characteristics and supporting evidence for translation and protein detection. This work will support future explorations of de novo genes and ORFs in humans

Improving mammalian genome scaffolding using large insert mate-pair next-generation sequencing

BACKGROUND: Paired-tag sequencing approaches are commonly used for the analysis of genome structure. However, mammalian genomes have a complex organization with a variety of repetitive elements that complicate comprehensive genome-wide analyses. RESULTS: Here, we systematically assessed the utility of paired-end and mate-pair (MP) next-generation sequencing libraries with insert sizes ranging from 170 bp to 25 kb, for genome coverage and for improving scaffolding of a mammalian genome (Rattus norvegicus). Despite a lower library complexity, large insert MP libraries (20 or 25 kb) provided very high physical genome coverage and were found to efficiently span repeat elements in the genome. Medium-sized (5, 8 or 15 kb) MP libraries were much more efficient for genome structure analysis than the more commonly used shorter insert paired-end and 3 kb MP libraries. Furthermore, the combination of medium- and large insert libraries resulted in a 3-fold increase in N50 in scaffolding processes. Finally, we show that our data can be used to evaluate and improve contig order and orientation in the current rat reference genome assembly. CONCLUSIONS: We conclude that applying combinations of mate-pair libraries with insert sizes that match the distributions of repetitive elements improves contig scaffolding and can contribute to the finishing of draft genomes

Systematic biases in DNA copy number originate from isolation procedures

BACKGROUND: The ability to accurately detect DNA copy number variation in both a sensitive and quantitative manner is important in many research areas. However, genome-wide DNA copy number analyses are complicated by variations in detection signal. RESULTS: While GC content has been used to correct for this, here we show that coverage biases are tissue-specific and independent of the detection method as demonstrated by next-generation sequencing and array CGH. Moreover, we show that DNA isolation stringency affects the degree of equimolar coverage and that the observed biases coincide with chromatin characteristics like gene expression, genomic isochores, and replication timing. CONCLUSION: These results indicate that chromatin organization is a main determinant for differential DNA retrieval. These findings are highly relevant for germline and somatic DNA copy number variation analyses

Translational control of cardiac fibrosis

Background Fibrosis is a common pathology in many cardiac disorders and is driven by the activation of resident fibroblasts. The global post-transcriptional mechanisms underlying fibroblast-to-myofibroblast conversion in the heart have not been explored. Methods Genome-wide changes of RNA transcription and translation during human cardiac fibroblast activation were monitored with RNA sequencing and ribosome profiling. We then used miRNA-and RNA-binding protein-based analyses to identify translational regulators of fibrogenic genes. To reveal post-transcriptional mechanisms in the human fibrotic heart, we then integrated our findings with cardiac ribosome occupancy levels of 30 dilated cardiomyopathy patients. Results We generated nucleotide-resolution translatome data during the TGFβ1-driven cellular transition of human cardiac fibroblasts to myofibroblasts. This identified dynamic changes of RNA transcription and translation at several time points during the fibrotic response, revealing transient and early-responder genes. Remarkably, about one-third of all changes in gene expression in activated fibroblasts are subject to translational regulation and dynamic variation in ribosome occupancy affects protein abundance independent of RNA levels. Targets of RNA-binding proteins were strongly enriched in post-transcriptionally regulated genes, suggesting genes such as MBNL2 can act as translational activators or repressors. Ribosome occupancy in the hearts of patients with dilated cardiomyopathy suggested an extensive post-transcriptional regulatory network underlying cardiac fibrosis. Key network hubs include RNA-binding proteins such as PUM2 and QKI that work in concert to regulate the translation of target transcripts in human diseased hearts. Conclusions We reveal widespread translational effects of TGFβ1 and define novel post-transcriptional events that control the fibroblast-to-myofibroblast transition. Regulatory networks that affect ribosome occupancy in fibroblasts are paralleled in human heart disease. Our findings show the central importance of translational control in fibrosis and highlight novel pathogenic mechanisms in heart failure

Targeting pediatric cancers via T-cell recognition of the monomorphic MHC class I-related protein MR1

Human leukocyte antigen (HLA) restriction of conventional T-cell targeting introduces complexity in generating T-cell therapy strategies for patients with cancer with diverse HLA-backgrounds. A subpopulation of atypical, major histocompatibility complex-I related protein 1 (MR1)-restricted T-cells, distinctive from mucosal-associated invariant T-cells (MAITs), was recently identified recognizing currently unidentified MR1-presented cancer-specific metabolites. It is hypothesized that the MC.7.G5 MR1T-clone has potential as a pan-cancer, pan-population T-cell immunotherapy approach. These cells are irresponsive to healthy tissue while conferring T-cell receptor(TCR) dependent, HLA-independent cytotoxicity to a wide range of adult cancers. Studies so far are limited to adult malignancies. Here, we investigated the potential of MR1-targeting cellular therapy strategies in pediatric cancer. Bulk RNA sequencing data of primary pediatric tumors were analyzed to assess MR1 expression. In vitro pediatric tumor models were subsequently screened to evaluate their susceptibility to engineered MC.7.G5 TCR-expressing T-cells. Targeting capacity was correlated with qPCR-based MR1 mRNA and protein overexpression. RNA expression of MR1 in primary pediatric tumors varied widely within and between tumor entities. Notably, embryonal tumors exhibited significantly lower MR1 expression than other pediatric tumors. In line with this, most screened embryonal tumors displayed resistance to MR1T-targeting in vitro MR1T susceptibility was observed particularly in pediatric leukemia and diffuse midline glioma models. This study demonstrates potential of MC.7.G5 MR1T-cell immunotherapy in pediatric leukemias and diffuse midline glioma, while activity against embryonal tumors was limited. The dismal prognosis associated with relapsed/refractory leukemias and high-grade brain tumors highlights the promise to improve survival rates of children with these cancers

Titin-truncating variants affect heart function in disease cohorts and the general population

Titin-truncating variants (TTNtv) commonly cause dilated cardiomyopathy (DCM). TTNtv are also encountered in ~1% of the general population, where they may be silent, perhaps reflecting allelic factors. To better understand TTNtv, we integrated TTN allelic series, cardiac imaging and genomic data in humans and studied rat models with disparate TTNtv. In patients with DCM, TTNtv throughout titin were significantly associated with DCM. Ribosomal profiling in rat showed the translational footprint of premature stop codons in Ttn, TTNtv-position-independent nonsense-mediated degradation of the mutant allele and a signature of perturbed cardiac metabolism. Heart physiology in rats with TTNtv was unremarkable at baseline but became impaired during cardiac stress. In healthy humans, machine-learning-based analysis of high-resolution cardiac imaging showed TTNtv to be associated with eccentric cardiac remodeling. These data show that TTNtv have molecular and physiological effects on the heart across species, with a continuum of expressivity in health and disease

Rattus norvegicus BN/SHR liver and heart left ventricle ribosomal RNA depleted directional RNA sequencing

Abstract Objective The spontaneously hypertensive rat strain is a frequently used disease model. In a previous study, we measured translational efficiency from this strain and BN-Lx animals. Here, we describe long RNA sequencing reads from ribosomal RNA depleted samples from the same animals. This data can be used to investigate splicing-related events. Results RNA was extracted from rat liver and heart left ventricle from BN-Lx and SHR/Ola rats in biological replicates. Ribosomal RNA was removed and the samples subjected to directional high-throughput RNA-sequencing. Read and alignment statistics indicate high quality of the data. The raw sequencing reads are freely available on the NCBI short read archive and can be used for further research on tissue and strain differences, or analysed together with other published high-throughput data from the same animals

Lack of Major Genome Instability in Tumors of p53 Null Rats

Tumorigenesis is often associated with loss of tumor suppressor genes (such as TP53), genomic instability and telomere lengthening. Previously, we generated and characterized a rat p53 knockout model in which the homozygous rats predominantly develop hemangiosarcomas whereas the heterozygous rats mainly develop osteosarcomas. Using genome-wide analyses, we find that the tumors that arise in the heterozygous and homozygous Tp53(C273X) mutant animals are also different in their genomic instability profiles. While p53 was fully inactivated in both heterozygous and homozygous knockout rats, tumors from homozygous animals show very limited aneuploidy and low degrees of somatic copy number variation as compared to the tumors from heterozygous animals. In addition, complex structural rearrangements such as chromothripsis and breakage-fusion-bridge cycles were never found in tumors from homozygous animals, while these were readily detectable in tumors from heterozygous animals. Finally, we measured telomere length and telomere lengthening pathway activity and found that tumors of homozygous animals have longer telomeres but do not show clear telomerase or alternative lengthening of telomeres (ALT) activity differences as compared to the tumors from heterozygous animals. Taken together, our results demonstrate that host p53 status in this rat p53 knockout model has a large effect on both tumor type and genomic instability characteristics, where full loss of functional p53 is not the main driver of large-scale structural variations. Our results also suggest that chromothripsis primarily occurs under p53 heterozygous rather than p53 null conditions

Lack of Major Genome Instability in Tumors of p53 Null Rats

Tumorigenesis is often associated with loss of tumor suppressor genes (such as TP53), genomic instability and telomere lengthening. Previously, we generated and characterized a rat p53 knockout model in which the homozygous rats predominantly develop hemangiosarcomas whereas the heterozygous rats mainly develop osteosarcomas. Using genome-wide analyses, we find that the tumors that arise in the heterozygous and homozygous Tp53(C273X) mutant animals are also different in their genomic instability profiles. While p53 was fully inactivated in both heterozygous and homozygous knockout rats, tumors from homozygous animals show very limited aneuploidy and low degrees of somatic copy number variation as compared to the tumors from heterozygous animals. In addition, complex structural rearrangements such as chromothripsis and breakage-fusion-bridge cycles were never found in tumors from homozygous animals, while these were readily detectable in tumors from heterozygous animals. Finally, we measured telomere length and telomere lengthening pathway activity and found that tumors of homozygous animals have longer telomeres but do not show clear telomerase or alternative lengthening of telomeres (ALT) activity differences as compared to the tumors from heterozygous animals. Taken together, our results demonstrate that host p53 status in this rat p53 knockout model has a large effect on both tumor type and genomic instability characteristics, where full loss of functional p53 is not the main driver of large-scale structural variations. Our results also suggest that chromothripsis primarily occurs under p53 heterozygous rather than p53 null conditions