PLOS Genetics

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ISSN / EISSN : 15537390 / 15537404
Current Publisher: Public Library of Science (PLoS) (10.1371)
Total articles ≅ 8,497
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Keemo Delos Santos, Minhee Kim, Christine Yergeau, Steve Jean, Nam-Sung Moon
Published: 5 September 2019
PLOS Genetics, Volume 15; doi:10.1371/journal.pgen.1008376

Abstract:Phosphoribosyl pyrophosphate synthetase (PRPS) is a rate-limiting enzyme whose function is important for the biosynthesis of purines, pyrimidines, and pyridines. Importantly, while missense mutations of PRPS1 have been identified in neurological disorders such as Arts syndrome, how they contribute to neuropathogenesis is still unclear. We identified the Drosophila ortholog of PRPS (dPRPS) as a direct target of RB/E2F in Drosophila, a vital cell cycle regulator, and engineered dPRPS alleles carrying patient-derived mutations. Interestingly, while they are able to develop normally, dPRPS mutant flies have a shortened lifespan and locomotive defects, common phenotypes associated with neurodegeneration. Careful analysis of the fat body revealed that patient-derived PRPS mutations result in profound defects in lipolysis, macroautophagy, and lysosome function. Significantly, we show evidence that the nervous system of dPRPS mutant flies is affected by these defects. Overall, we uncovered an unexpected link between nucleotide metabolism and autophagy/lysosome function, providing a possible mechanism by which PRPS-dysfunction contributes to neurological disorders. Phosphoribosyl pyrophosphate synthetase (PRPS) is an important enzyme in nucleotide synthesis: the building blocks of DNA and RNA and other important metabolites. Importantly, while PRPS is mutated in neurological disorders such as Arts syndrome, Charcot-Marie-Tooth disease, and nonsyndromic sensorineural deafness, it is currently unclear why PRPS dysfunction leads to neurological disorders. In this study, we engineered a Drosophila model of ARTS syndrome and discovered that PRPS mutations result in defects in lysosome-mediated and autophagy processes, which are known to be important for neuronal homeostasis. Our study provides crucial insights into the way that PRPS mutations contribute to neurological disorders.
Linda Anderegg, Michelle Im Hof Gut, Udo Hetzel, Elizabeth W. Howerth, Fabienne Leuthard, Kaisa Kyöstilä, Hannes Lohi, Louise Pettitt, Cathryn Mellersh, Katie M. Minor, et al.
Published: 3 September 2019
PLOS Genetics, Volume 15; doi:10.1371/journal.pgen.1008378

Abstract:Primary ciliary dyskinesia (PCD) is a hereditary defect of motile cilia in humans and several domestic animal species. Typical clinical findings are chronic recurrent infections of the respiratory tract and fertility problems. We analyzed an Alaskan Malamute family, in which two out of six puppies were affected by PCD. The parents were unaffected suggesting autosomal recessive inheritance. Linkage and homozygosity mapping defined critical intervals comprising ~118 Mb. Whole genome sequencing of one case and comparison to 601 control genomes identified a disease associated frameshift variant, c.43delA, in the NME5 gene encoding a sparsely characterized protein associated with ciliary function. Nme5-/- knockout mice exhibit doming of the skull, hydrocephalus and sperm flagellar defects. The genotypes at NME5:c.43delA showed the expected co-segregation with the phenotype in the Alaskan Malamute family. An additional unrelated Alaskan Malamute with PCD and hydrocephalus that became available later in the study was also homozygous mutant at the NME5:c.43delA variant. The mutant allele was not present in more than 1000 control dogs from different breeds. Immunohistochemistry demonstrated absence of the NME5 protein from nasal epithelia of an affected dog. We therefore propose NME5:c.43delA as the most likely candidate causative variant for PCD in Alaskan Malamutes. These findings enable genetic testing to avoid the unintentional breeding of affected dogs in the future. Furthermore, the results of this study identify NME5 as a novel candidate gene for unsolved human PCD and/or hydrocephalus cases. Motile cilia are required for clearing mucous, infectious agents and inhaled dust from the airways. Primary ciliary dyskinesia (PCD) is a hereditary defect of motile cilia. Clinical findings may include recurrent airway infections, fertility problems, and sometimes hydrocephalus. We analyzed an Alaskan Malamute family, in which two out of six puppies were affected by an autosomal recessive form of PCD. Whole genome sequencing of an affected dog identified a one base pair deletion in the NME5 gene, c.43delA, leading to an early frame-shift and premature stop codon. Later in the study, we became aware of a previously published Alaskan Malamute with PCD involving respiratory infections and hydrocephalus. We observed perfect concordance of the NME5 genotypes with the PCD phenotype in all three affected Alaskan Malamutes and more than 1000 controls. The fact that the third case, which had no documented close relationship to the initial two cases, was homozygous for the same rare mutant NME5 allele, strongly supports our hypothesis that NME5:c.43delA causes the PCD phenotype. We confirmed absence of NME5 protein expression in nasal epithelium of an affected dog. Our results enable genetic testing in dogs and identify NME5 as novel candidate gene for unsolved human PCD cases.
Alexander Kozik, Beth A. Rowan, Dean Lavelle, Lidija Berke, M. Eric Schranz, Richard W. Michelmore, Alan C. Christensen
Published: 30 August 2019
PLOS Genetics, Volume 15; doi:10.1371/journal.pgen.1008373

Abstract:Plant mitochondrial genomes are usually assembled and displayed as circular maps based on the widely-held view across the broad community of life scientists that circular genome-sized molecules are the primary form of plant mitochondrial DNA, despite the understanding by plant mitochondrial researchers that this is an inaccurate and outdated concept. Many plant mitochondrial genomes have one or more pairs of large repeats that can act as sites for inter- or intramolecular recombination, leading to multiple alternative arrangements (isoforms). Most mitochondrial genomes have been assembled using methods unable to capture the complete spectrum of isoforms within a species, leading to an incomplete inference of their structure and recombinational activity. To document and investigate underlying reasons for structural diversity in plant mitochondrial DNA, we used long-read (PacBio) and short-read (Illumina) sequencing data to assemble and compare mitochondrial genomes of domesticated (Lactuca sativa) and wild (L. saligna and L. serriola) lettuce species. We characterized a comprehensive, complex set of isoforms within each species and compared genome structures between species. Physical analysis of L. sativa mtDNA molecules by fluorescence microscopy revealed a variety of linear, branched, and circular structures. The mitochondrial genomes for L. sativa and L. serriola were identical in sequence and arrangement and differed substantially from L. saligna, indicating that the mitochondrial genome structure did not change during domestication. From the isoforms in our data, we infer that recombination occurs at repeats of all sizes at variable frequencies. The differences in genome structure between L. saligna and the two other Lactuca species can be largely explained by rare recombination events that rearranged the structure. Our data demonstrate that representations of plant mitochondrial genomes as simple, circular molecules are not accurate descriptions of their true nature and that in reality plant mitochondrial DNA is a complex, dynamic mixture of forms. Plant mitochondrial genomes are commonly depicted in research articles and textbooks as circular molecules that are the size of the genome. Although research on mitochondrial DNA (mtDNA) over the past few decades has revealed that genome-sized circles are exceedingly rare and that alternative forms of mtDNA are more common, many biologists still perceive circular maps as representing one or more physical chromosomes. This misconception can lead to biases in how mitochondrial genomes are assembled and misinterpretation of their evolutionary relationships, synteny, and histories. In this study, we present an assembly methodology that uses short- and long-read sequencing data to determine the mitochondrial genome structures of three lettuce species. We show that these mitochondrial genomes are fluid and dynamic, with multiple sequence arrangements of the genome coexisting within individuals of...
Shaoke Lou, Kellie A. Cotter, Tianxiao Li, Jin Liang, Hussein Mohsen, Jason Liu, Jing Zhang, Sandra Cohen, Jinrui Xu, Haiyuan Yu, et al.
Published: 30 August 2019
PLOS Genetics, Volume 15; doi:10.1371/journal.pgen.1007860

Abstract:There has been much effort to prioritize genomic variants with respect to their impact on “function”. However, function is often not precisely defined: sometimes it is the disease association of a variant; on other occasions, it reflects a molecular effect on transcription or epigenetics. Here, we coupled multiple genomic predictors to build GRAM, a GeneRAlized Model, to predict a well-defined experimental target: the expression-modulating effect of a non-coding variant on its associated gene, in a transferable, cell-specific manner. Firstly, we performed feature engineering: using LASSO, a regularized linear model, we found transcription factor (TF) binding most predictive, especially for TFs that are hubs in the regulatory network; in contrast, evolutionary conservation, a popular feature in many other variant-impact predictors, has almost no contribution. Moreover, TF binding inferred from in vitro SELEX is as effective as that from in vivo ChIP-Seq. Second, we implemented GRAM integrating only SELEX features and expression profiles; thus, the program combines a universal regulatory score with an easily obtainable modifier reflecting the particular cell type. We benchmarked GRAM on large-scale MPRA datasets, achieving AUROC scores of 0.72 in GM12878 and 0.66 in a multi-cell line dataset. We then evaluated the performance of GRAM on targeted regions using luciferase assays in the MCF7 and K562 cell lines. We noted that changing the insertion position of the construct relative to the reporter gene gave very different results, highlighting the importance of carefully defining the exact prediction target of the model. Finally, we illustrated the utility of GRAM in fine-mapping causal variants and developed a practical software pipeline to carry this out. In particular, we demonstrated in specific examples how the pipeline could pinpoint variants that directly modulate gene expression within a larger linkage-disequilibrium block associated with a phenotype of interest (e.g., for an eQTL). With advances in sequencing technologies, a deluge of genomic data is available; however, only a fraction of non-coding genomic variants are functionally relevant. Sifting through this data to prioritize genomic variants with respect to function is an important but challenging task. In this study, we built GRAM, a GeneRAlized Model, to predict the expression-modulating effects of non-coding variants in a cell-specific manner. GRAM combines a universal regulatory score defined by transcription factor binding with an easily obtainable modifier defined by transcription factor binding and expression to reflect the particular cell type. We evaluated this framework on multiple cell lines with high performance and showed that it could be applied to any cell line or sample with gene expression data. We also integrated GRAM into a practical software pipeline to fine-map causal variants that directly modulate gene expression among a larger linkage-disequilibrium...
Cavin Wong, Fei Chen, Najmeh Alirezaie, Yifan Wang, Adeline Cuggia, Ayelet Borgida, Spring Holter, Tatiana Lenko, Celine Domecq, Gloria M. Petersen, et al.
Published: 30 August 2019
PLOS Genetics, Volume 15; doi:10.1371/journal.pgen.1008344

Abstract:Pancreatic adenocarcinoma (PC) is a lethal malignancy that is familial or associated with genetic syndromes in 10% of cases. Gene-based surveillance strategies for at-risk individuals may improve clinical outcomes. However, familial PC (FPC) is plagued by genetic heterogeneity and the genetic basis for the majority of FPC remains elusive, hampering the development of gene-based surveillance programs. The study was powered to identify genes with a cumulative pathogenic variant prevalence of at least 3%, which includes the most prevalent PC susceptibility gene, BRCA2. Since the majority of known PC susceptibility genes are involved in DNA repair, we focused on genes implicated in these pathways. We performed a region-based association study using the Mixed-Effects Score Test, followed by leave-one-out characterization of PC-associated gene regions and variants to identify the genes and variants driving risk associations. We evaluated 398 cases from two case series and 987 controls without a personal history of cancer. The first case series consisted of 109 patients with either FPC (n = 101) or PC at ≤50 years of age (n = 8). The second case series was composed of 289 unselected PC cases. We validated this discovery strategy by identifying known pathogenic BRCA2 variants, and also identified SMG1, encoding a serine/threonine protein kinase, to be significantly associated with PC following correction for multiple testing (p = 3.22x10-7). The SMG1 association was validated in a second independent series of 532 FPC cases and 753 controls (pG SMG1 variant in 3 affected relatives in a FPC kindred, and we found c.103G>A to be a recurrent SMG1 variant associating with PC in both the discovery and validation series. These results suggest that SMG1 is a novel PC susceptibility gene, and we identified specific SMG1 gene variants associated with PC risk. Pancreatic cancer (PC) remains one of the most lethal malignancies, with a 5-year survival rate of only 8%. Approximately 10% of PC occurs in families or in patients with hereditary mutations that are known to cause PC. The genetic causes of familial PC remain largely unknown. Using a new statistical method, we tested 398 patients with PC and 987 individuals without cancer (discovery series) to identify hereditary genetic variabilities that associate with PC. As a proof of principle for our methodology, we identified mutations in the BRCA2 gene, which is known to cause PC. We also identified SMG1 as a novel gene that associates with PC risk. To support this finding, we confirmed our observations in a separate group of 532 patients with PC and 753 individuals without cancer (validation series). In addition, we provide additional genetic evidence to support our findings by showing that a SMG1 genetic change is present in three relatives with PC in a family. We also identified a recurrent SMG1 variant that associated with PC in both the discovery and validation series. Our observations suggest that SMG1 is...
Jingyuan Fu, Folkert Kuipers
Published: 29 August 2019
PLOS Genetics, Volume 15; doi:10.1371/journal.pgen.1008307

Julia H. Kemis, Vanessa Linke, Kelsey L. Barrett, Frederick J. Boehm, Lindsay L. Traeger, Mark P. Keller, Mary E. Rabaglia, Kathryn L. Schueler, Donald S. Stapleton, Daniel M. Gatti, et al.
Published: 29 August 2019
PLOS Genetics, Volume 15; doi:10.1371/journal.pgen.1008073

Abstract:The microbial communities that inhabit the distal gut of humans and other mammals exhibit large inter-individual variation. While host genetics is a known factor that influences gut microbiota composition, the mechanisms underlying this variation remain largely unknown. Bile acids (BAs) are hormones that are produced by the host and chemically modified by gut bacteria. BAs serve as environmental cues and nutrients to microbes, but they can also have antibacterial effects. We hypothesized that host genetic variation in BA metabolism and homeostasis influence gut microbiota composition. To address this, we used the Diversity Outbred (DO) stock, a population of genetically distinct mice derived from eight founder strains. We characterized the fecal microbiota composition and plasma and cecal BA profiles from 400 DO mice maintained on a high-fat high-sucrose diet for ~22 weeks. Using quantitative trait locus (QTL) analysis, we identified several genomic regions associated with variations in both bacterial and BA profiles. Notably, we found overlapping QTL for Turicibacter sp. and plasma cholic acid, which mapped to a locus containing the gene for the ileal bile acid transporter, Slc10a2. Mediation analysis and subsequent follow-up validation experiments suggest that differences in Slc10a2 gene expression associated with the different strains influences levels of both traits and revealed novel interactions between Turicibacter and BAs. This work illustrates how systems genetics can be utilized to generate testable hypotheses and provide insight into host-microbe interactions.
Hongliang Wang, Siyi Guo, Xin Qiao, Jianfei Guo, Zuliang Li, Yusen Zhou, Shenglong Bai, Zhiyong Gao, Daojie Wang, Pengcheng Wang, et al.
Published: 29 August 2019
PLOS Genetics, Volume 15; doi:10.1371/journal.pgen.1008377

Abstract:Intercellular communication in adjacent cell layers determines cell fate and polarity, thus orchestrating tissue specification and differentiation. Here we use the maize stomatal apparatus as a model to investigate cell fate determination. Mutations in ZmBZU2 (bizui2, bzu2) confer a complete absence of subsidiary cells (SCs) and normal guard cells (GCs), leading to failure of formation of mature stomatal complexes. Nuclear polarization and actin accumulation at the interface between subsidiary mother cells (SMCs) and guard mother cells (GMCs), an essential pre-requisite for asymmetric cell division, did not occur in Zmbzu2 mutants. ZmBZU2 encodes a basic helix-loop-helix (bHLH) transcription factor, which is an ortholog of AtMUTE in Arabidopsis (BZU2/ZmMUTE). We found that a number of genes implicated in stomatal development are transcriptionally regulated by BZU2/ZmMUTE. In particular, BZU2/ZmMUTE directly binds to the promoters of PAN1 and PAN2, two early regulators of protodermal cell fate and SMC polarization, consistent with the low levels of transcription of these genes observed in bzu2-1 mutants. BZU2/ZmMUTE has the cell-to-cell mobility characteristic similar to that of BdMUTE in Brachypodium distachyon. Unexpectedly, BZU2/ZmMUTE is expressed in GMC from the asymmetric division stage to the GMC division stage, and especially in the SMC establishment stage. Taken together, these data imply that BZU2/ZmMUTE is required for early events in SMC polarization and differentiation as well as for the last symmetrical division of GMCs to produce the two GCs, and is a master determinant of the cell fate of its neighbors through cell-to-cell communication. In the grasses, individual stomatal complexes comprise a pair of dumbbell-shaped guard cells associated with two subsidiary cells and the pore, which together play essential roles in the exchange of CO2 and O2, in xylem transport, and in transpiration. However, little is known about grass stomatal complex development. We have uncovered and characterized a key factor (BZU2/ZmMUTE) determining the formation of guard cells in Zea mays. Our data suggest that BZU2/ZmMUTE has a dual role, both as an important player in determining the formation of the guard mother cell, as well as being required for polarization and recruitment of the subsidiary mother cells.
Alison Wood, Sophie E. Irving, Daniel J. Bennison, Rebecca M. Corrigan
Published: 29 August 2019
PLOS Genetics, Volume 15; doi:10.1371/journal.pgen.1008346

Abstract:Ribosome assembly cofactors are widely conserved across all domains of life. One such group, the ribosome-associated GTPases (RA-GTPase), act as molecular switches to coordinate ribosome assembly. We previously identified the Staphylococcus aureus RA-GTPase Era as a target for the stringent response alarmone (p)ppGpp, with binding leading to inhibition of GTPase activity. Era is highly conserved throughout the bacterial kingdom and is essential in many species, although the function of Era in ribosome assembly is unclear. Here we show that Era is not essential in S. aureus but is important for 30S ribosomal subunit assembly. Protein interaction studies reveal that Era interacts with the 16S rRNA endonuclease YbeY and the DEAD-box RNA helicase CshA. We determine that both Era and CshA are required for growth at suboptimal temperatures and rRNA processing. Era and CshA also form direct interactions with the (p)ppGpp synthetase RelSau, with RelSau positively impacting the GTPase activity of Era but negatively affecting the helicase activity of CshA. We propose that in its GTP-bound form, Era acts as a hub protein on the ribosome to direct enzymes involved in rRNA processing/degradation and ribosome subunit assembly to their site of action. This activity is impeded by multiple components of the stringent response, contributing to the slowed growth phenotype synonymous with this stress response pathway. The bacterial ribosome is an essential cellular component and as such is the target for a number of currently used antimicrobials. Correct assembly of this complex macromolecule requires a number of accessory enzymes, the functions of which are poorly characterised. Here we examine the function of Era, a GTPase enzyme involved in 30S ribosomal subunit biogenesis in the important human pathogen S. aureus. We uncover that Era is not an essential enzyme in S. aureus, as it is in many other species, but is important for correct ribosome assembly. In a bid to determine a function for this enzyme in ribosomal assembly, we identify a number of protein interaction partners with roles in ribosomal RNA maturation or degradation, supporting the idea that Era acts as a hub protein facilitating ribosomal biogenesis. We also uncover a link between Era and the (p)ppGpp synthetase RelSau, revealing an additional level of control of rRNA processing by the stringent response. With this study we elaborate on the functions of GTPases in ribosomal assembly, processes that are controlled at multiple points by the stringent response.
Aubin Fleiss, Samuel O'donnell, Téo Fournier, Wenqing Lu, Nicolas Agier, Stéphane Delmas, Joseph Schacherer, Gilles Fischer
Published: 29 August 2019
PLOS Genetics, Volume 15; doi:10.1371/journal.pgen.1008332

Abstract:Genome engineering is a powerful approach to study how chromosomal architecture impacts phenotypes. However, quantifying the fitness impact of translocations independently from the confounding effect of base substitutions has so far remained challenging. We report a novel application of the CRISPR/Cas9 technology allowing to generate with high efficiency both uniquely targeted and multiple concomitant reciprocal translocations in the yeast genome. Targeted translocations are constructed by inducing two double-strand breaks on different chromosomes and forcing the trans-chromosomal repair through homologous recombination by chimerical donor DNAs. Multiple translocations are generated from the induction of several DSBs in LTR repeated sequences and promoting repair using endogenous uncut LTR copies as template. All engineered translocations are markerless and scarless. Targeted translocations are produced at base pair resolution and can be sequentially generated one after the other. Multiple translocations result in a large diversity of karyotypes and are associated in many instances with the formation of unanticipated segmental duplications. To test the phenotypic impact of translocations, we first recapitulated in a lab strain the SSU1/ECM34 translocation providing increased sulphite resistance to wine isolates. Surprisingly, the same translocation in a laboratory strain resulted in decreased sulphite resistance. However, adding the repeated sequences that are present in the SSU1 promoter of the resistant wine strain induced sulphite resistance in the lab strain, yet to a lower level than that of the wine isolate, implying that additional polymorphisms also contribute to the phenotype. These findings illustrate the advantage brought by our technique to untangle the phenotypic impacts of structural variations from confounding effects of base substitutions. Secondly, we showed that strains with multiple translocations, even those devoid of unanticipated segmental duplications, display large phenotypic diversity in a wide range of environmental conditions, showing that simply reconfiguring chromosome architecture is sufficient to provide fitness advantages in stressful growth conditions. Chromosomes are highly dynamic objects that often undergo large structural variations such as reciprocal translocations. Such rearrangements can have dramatic functional consequences, as they can disrupt genes, change their regulation or create novel fusion genes at their breakpoints. For instance, 90–95% of patients diagnosed with chronic myeloid leukemia carry the Philadelphia chromosome characterized by a reciprocal translocation between chromosomes 9 and 22. In addition, translocations reorganize the genetic information along chromosomes, which in turn can modify the 3D architecture of the genome and potentially affect its functioning. Quantifying the fitness impact of translocations independently from the confounding effect of base substitutions has so...