Most Pleiotropic Effects of Gene Knockouts Are Evolutionarily Transient in Yeasts.

Pleiotropy, the phenomenon in which a single gene influences multiple traits, is a fundamental concept in genetics. However, the evolutionary mechanisms underlying pleiotropy require further investigation. In this study, we conducted parallel gene knockouts targeting 100 transcription factors in 2 strains of Saccharomyces cerevisiae. We systematically examined and quantified the pleiotropic effects of these knockouts on gene expression levels for each transcription factor. Our results showed that the knockout of a single gene generally affected the expression levels of multiple genes in both strains, indicating various degrees of pleiotropic effects. Strikingly, the pleiotropic effects of the knockouts change rapidly between strains in different genetic backgrounds, and ∼85% of them were nonconserved. Further analysis revealed that the conserved effects tended to be functionally associated with the deleted transcription factors, while the nonconserved effects appeared to be more ad hoc responses. In addition, we measured 184 yeast cell morphological traits in these knockouts and found consistent patterns. In order to investigate the evolutionary processes underlying pleiotropy, we examined the pleiotropic effects of standing genetic variations in a population consisting of ∼1,000 hybrid progenies of the 2 strains. We observed that newly evolved expression quantitative trait loci impacted the expression of a greater number of genes than did old expression quantitative trait loci, suggesting that natural selection is gradually eliminating maladaptive or slightly deleterious pleiotropic responses. Overall, our results show that, although being prevalent for new mutations, the majority of pleiotropic effects observed are evolutionarily transient, which explains how evolution proceeds despite complicated pleiotropic effects.

Immune profiling of adeno-associated virus response identifies B cell-specific targets that enable vector re-administration in mice.

Adeno-associated virus (AAV) vector-based gene therapies can be applied to a wide range of diseases. AAV expression can last for months to years, but vector re-administration may be necessary to achieve life-long treatment. Unfortunately, immune responses against these vectors are potentiated after the first administration, preventing the clinical use of repeated administration of AAVs. Reducing the immune response against AAVs while minimizing broad immunosuppression would improve gene delivery efficiency and long-term safety. In this study, we quantified the contributions of multiple immune system components of the anti-AAV response in mice. We identified B-cell-mediated immunity as a critical component preventing vector re-administration. Additionally, we found that IgG depletion alone was insufficient to enable re-administration, suggesting IgM antibodies play an important role in the immune response against AAV. Further, we found that AAV-mediated transduction is improved in µMT mice that lack functional IgM heavy chains and cannot form mature B-cells relative to wild-type mice. Combined, our results suggest that B-cells, including non-class switched B-cells, are a potential target for therapeutics enabling AAV re-administration. Our results also suggest that the µMT mice are a potentially useful experimental model for gene delivery studies since they allow repeated dosing for more efficient gene delivery from AAVs.

ARID2, a rare cause of Coffin-Siris syndrome: A novel microdeletion at 12q12q13.11 causing severe short stature and literature review.

Coffin-Siris syndrome (CSS) 6 is caused by heterozygous pathogenic variants in the AT-rich interaction domain 2 (ARID2) gene on 12q12. Currently, only 26 cases with both detailed clinical and genetic information have been documented in the literature. Microdeletions of the entire ARID2 gene are rare. In this study, we report a 5-year-7-month-old Chinese female who underwent whole-exome sequencing to discover that she had a de novo 1.563 Mb heterozygous copy number loss at 12q12q13.11, involving an entire deletion of ARID2. The female had severe short stature with obvious dysmorphic facial features, global developmental delay and hypoplastic fingers and toes. Her growth hormone level was normal, with reduced IGF-1 and increased CA19-9 levels. After a review of the 27 patients with ARID2 deficiency, a significant positive correlation was observed between age and height standard deviation score (SDS) (r = 0.71, p = 0.0002), suggesting a possibility of growth catch-up. This study expands the genetic and phenotypic spectrum of CCS6 and provides a decision-making reference for growth hormone therapy.

Transcriptome Analysis in Yeast Reveals the Externality of Position Effects.

The activity of a gene newly integrated into a chromosome depends on the genomic context of the integration site. This "position effect" has been widely reported, although the other side of the coin, that is, how integration affects the local chromosomal environment, has remained largely unexplored, as have the mechanism and phenotypic consequences of this "externality" of the position effect. Here, we examined the transcriptome profiles of approximately 250 Saccharomyces cerevisiae strains, each with GFP integrated into a different locus of the wild-type strain. We found that in genomic regions enriched in essential genes, GFP expression tended to be lower, and the genes near the integration site tended to show greater expression reduction. Further joint analysis with public genome-wide histone modification profiles indicated that this effect was associated with H3K4me2. More importantly, we found that changes in the expression of neighboring genes, but not GFP expression, significantly altered the cellular growth rate. As a result, genomic loci that showed high GFP expression immediately after integration were associated with growth disadvantages caused by elevated expression of neighboring genes, ultimately leading to a low total yield of GFP in the long run. Our results were consistent with competition for transcriptional resources among neighboring genes and revealed a previously unappreciated facet of position effects. This study highlights the impact of position effects on the fate of exogenous gene integration and has significant implications for biological engineering and the pathology of viral integration into the host genome.

Second Human Pegivirus in Hepatitis C Virus-Infected and Hepatitis C Virus/HIV-1-Co-infected Persons Who Inject Drugs, China.

We report the presence of the second human pegivirus (HPgV-2) in Guangdong and Sichuan Provinces in China. The prevalence of HPgV-2 in hepatitis C virus/HIV-1-co-infected persons who inject drugs was 12.9% in Guangdong and 15.9% in Sichuan. This population is at high risk for HPgV-2 infection.

An RNA-sequencing transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex.

The major cell classes of the brain differ in their developmental processes, metabolism, signaling, and function. To better understand the functions and interactions of the cell types that comprise these classes, we acutely purified representative populations of neurons, astrocytes, oligodendrocyte precursor cells, newly formed oligodendrocytes, myelinating oligodendrocytes, microglia, endothelial cells, and pericytes from mouse cerebral cortex. We generated a transcriptome database for these eight cell types by RNA sequencing and used a sensitive algorithm to detect alternative splicing events in each cell type. Bioinformatic analyses identified thousands of new cell type-enriched genes and splicing isoforms that will provide novel markers for cell identification, tools for genetic manipulation, and insights into the biology of the brain. For example, our data provide clues as to how neurons and astrocytes differ in their ability to dynamically regulate glycolytic flux and lactate generation attributable to unique splicing of PKM2, the gene encoding the glycolytic enzyme pyruvate kinase. This dataset will provide a powerful new resource for understanding the development and function of the brain. To ensure the widespread distribution of these datasets, we have created a user-friendly website (http://web.stanford.edu/group/barres_lab/brain_rnaseq.html) that provides a platform for analyzing and comparing transciption and alternative splicing profiles for various cell classes in the brain.

Genome-wide probing of eukaryotic nascent RNA structure elucidates cotranscriptional folding and its antimutagenic effect.

The transcriptional intermediates of RNAs fold into secondary structures with multiple regulatory roles, yet the details of such cotranscriptional RNA folding are largely unresolved in eukaryotes. Here, we present eSPET-seq (Structural Probing of Elongating Transcripts in eukaryotes), a method to assess the cotranscriptional RNA folding in Saccharomyces cerevisiae. Our study reveals pervasive structural transitions during cotranscriptional folding and overall structural similarities between nascent and mature RNAs. Furthermore, a combined analysis with genome-wide R-loop and mutation rate approximations provides quantitative evidence for the antimutator effect of nascent RNA folding through competitive inhibition of the R-loops, known to facilitate transcription-associated mutagenesis. Taken together, we present an experimental evaluation of cotranscriptional folding in eukaryotes and demonstrate the antimutator effect of nascent RNA folding. These results suggest genome-wide coupling between the processing and transmission of genetic information through RNA folding.

Description of the molecular and phenotypic spectrum in Chinese patients with aggrecan deficiency: Novel ACAN heterozygous variants in eight Chinese children and a review of the literature.

Objective: This study analyzed eight Chinese short stature children with aggrecan deficiency, and aimed to investigate potential genotype-phenotype correlations, differences in clinical characteristics between the Chinese and the Western populations, and effectiveness of recombinant human growth hormone therapy in patients with ACAN variants through a review of the literature. Methods: Pediatric short stature patients with ACAN heterozygous variants were identified using whole-exome sequencing. Subsequently, a literature review was carried out to summarize the clinical features, genetic findings, and efficacy of growth-promoting therapy in patients with ACAN variants. Results: We identified seven novel ACAN mutations and one recurrent variant. Patients in our center manifested with short stature (average height SDS: -3.30 ± 0.85) with slight dysmorphic characteristics. The prevalence of dysmorphic features in the Chinese populations is significantly lower than that in the Western populations. Meanwhile, only 24.24% of aggrecan-deficient Chinese children showed significantly advanced bone age (BA). Promising therapeutic benefits were seen in the patients who received growth-promoting treatment, with an increase in growth velocity from 4.52 ± 1.00 cm/year to 8.03 ± 1.16 cm/year. Conclusion: This study further expanded the variation spectrum of the ACAN gene and demonstrated that Chinese children with short stature who carried ACAN heterozygous variants exhibited early growth cessation, which may remain unnoticed by clinicians as most of these children had very mild dysmorphic characteristics and showed BA that was consistent with the chronological age. Genetic testing may help in the diagnosis.

Adeno-Associated Virus as Gene Delivery Vehicle into the Retina.

Initially discovered as a contaminant of adenovirus preparations, adeno-associated virus (AAV) has proved one of the most promising viral vectors for human gene therapy. The safety profile of AAV has been well-characterized in vivo studies, and the first gene therapy for patients with vision loss caused by Leber congenital amaurosis or retinitis pigmentosa was approved by the US Food and Drug Administration in 2017. This is an exciting era for investigators working on retina biology and treatments for blindness. In this chapter, we provide detailed methods for laboratory-scale production, purification, and characterization of AAV.

Dissimilation of synonymous codon usage bias in virus-host coevolution due to translational selection.

Eighteen of the 20 amino acids are each encoded by more than one synonymous codon. Due to differential transfer RNA supply within the cell, synonymous codons are not used with equal frequency, a phenomenon termed codon usage bias (CUB). Previous studies have demonstrated that CUB of endogenous genes trans-regulates the translational efficiency of other genes. We hypothesized similar effects for CUB of exogenous genes on host translation, and tested it in the case of viral infection, a common form of naturally occurring exogenous gene translation. We analysed public Ribo-Seq datasets from virus-infected yeast and human cells and showed that virus CUB trans-regulated tRNA availability, and therefore the relative decoding time of codons. Manipulative experiments in yeast using 37 synonymous fluorescent proteins confirmed that an exogenous gene with CUB more similar to that of the host would apply decreased translational load on the host per unit of expression, whereas expression of the exogenous gene was elevated. The combination of these two effects was that exogenous genes with CUB overly similar to that of the host severely impeded host translation. Finally, using a manually curated list of viruses and natural and symptomatic hosts, we found that virus CUB tended to be more similar to that of symptomatic hosts than that of natural hosts, supporting a general deleterious effect of excessive CUB similarity between virus and host. Our work revealed repulsion between virus and host CUBs when they are overly similar, a previously unrecognized complexity in the coevolution of virus and host.

Characterization of Humoral Immune Responses against Capsid Protein p24 and Transmembrane Glycoprotein gp41 of Human Immunodeficiency Virus Type 1 in China.

The objective of this study was to extend our previous research and to further characterize the humoral immune responses against HIV-1 p24, gp41 and the specific peptides carrying the immunodominant epitopes (IDEs) that react with human serum samples from HIV-1-infected individuals in China. We found that the majority (90.45%, 180/199) of the samples did not react with any of the three HIV-1 p24 peptides carrying IDEs, but did react with the recombinant full-length p24, suggesting that these samples tested in China were primarily directed against the conformational epitopes of HIV-1 p24. In contrast, 84.54% (164/194) of the samples reacted with at least one HIV-1 linear gp41 peptide, in particular the gp41-p1 peptide (amino acids 560-616). Both recently and long-term HIV-1-infected individuals displayed similar humoral immune responses against the recombinant gp41. However, samples from long-term HIV-1-infected subjects but not from recently infected subjects, showed a very strong reaction against the gp41-p1 peptide. The different response patterns observed for the two groups against the gp41 and the peptide gp41-p1 were statistically significant (P<0.01, Chi-square test). These results have direct relevance and importance for design of improved HIV-1 p24 detection assays and the gp41- based immunoassay that can be used to reliably distinguish recent and long-term HIV-1 infection.

Identification of key factors regulating self-renewal and differentiation in EML hematopoietic precursor cells by RNA-sequencing analysis.

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient's hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study. RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment. In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro and in vivo.

RNA-seq characterization of spinal cord injury transcriptome in acute/subacute phases: a resource for understanding the pathology at the systems level.

Spinal cord injury (SCI) is a devastating neurological disease without effective treatment. To generate a comprehensive view of the mechanisms involved in SCI pathology, we applied RNA-Sequencing (RNA-Seq) technology to characterize the temporal changes in global gene expression after contusive SCI in mice. We sequenced tissue samples from acute and subacute phases (2 days and 7 days after injury) and systematically characterized the transcriptomes with the goal of identifying pathways and genes critical in SCI pathology. The top enriched functional categories include "inflammation response," "neurological disease," "cell death and survival" and "nervous system development." The top enriched pathways include LXR/RXR Activation and Atherosclerosis Signaling, etc. Furthermore, we developed a systems-based analysis framework in order to identify key determinants in the global gene networks of the acute and sub-acute phases. Some candidate genes that we identified have been shown to play important roles in SCI, which demonstrates the validity of our approach. There are also many genes whose functions in SCI have not been well studied and can be further investigated by future experiments. We have also incorporated pharmacogenomic information into our analyses. Among the genes identified, the ones with existing drug information can be readily tested in SCI animal models. Therefore, in this study we have described an example of how global gene profiling can be translated to identifying genes of interest for functional tests in the future and generating new hypotheses. Additionally, the RNA-Seq enables splicing isoform identification and the estimation of expression levels, thus providing useful information for increasing the specificity of drug design and reducing potential side effect. In summary, these results provide a valuable reference data resource for a better understanding of the SCI process in the acute and sub-acute phases.