Investigate Gene Function Research Articles

Silencing of heat shock protein 90 (hsp90): Effect on development and infectivity of Ichthyophthirius multifiliis

BackgroundRecently, an increasing number of ichthyophthiriasis outbreaks has been reported, leading to high economic losses in fisheries and aquaculture. Although several strategies, including chemotherapeutics and immunoprophylaxis, have been implemented to control the parasite, no effective method is available. Hence, it is crucial to discover novel drug targets and vaccine candidates against Ichthyophthirius multifiliis. For this reason, understanding the parasite stage biology, host–pathogen interactions, molecular factors, regulation of major aspects during the invasion, and signaling pathways of the parasite can promote further prospects for disease management. Unfortunately, functional studies have been hampered in this ciliate due to the lack of robust methods for efficient nucleic acid delivery and genetic manipulation. In the current study, we used antisense technology to investigate the effects of targeted gene knockdown on the development and infectivity of I. multifiliis. Antisense oligonucleotides (ASOs) and their gold nanoconjugates were used to silence the heat shock protein 90 (hsp90) of I. multifiliis. Parasite stages were monitored for motility and development. In addition, the ability of the treated parasites to infect fish and cause disease was evaluated.ResultsWe demonstrated that ASOs were rapidly internalized by I. multifiliis and distributed diffusely throughout the cytosol. Knocking down of I. multifiliis hsp90 dramatically limited the growth and development of the parasite. In vivo exposure of common carp (Cyprinus carpio) showed reduced infectivity of ASO-treated theronts compared with the control group. No mortalities were recorded in the fish groups exposed to theronts pre-treated with ASOs compared with the 100% mortality observed in the non-treated control fish.ConclusionThis study presents a gene regulation approach for investigating gene function in I. multifiliis in vitro. In addition, we provide genetic evidence for the crucial role of hsp90 in the growth and development of the parasite, suggesting hsp90 as a novel therapeutic target for successful disease management. Further, this study introduces a useful tool and provides a significant contribution to the assessing and understanding of gene function in I. multifiliis.

Agrobacterium tumefaciens-Mediated Genetic Transformation of Narrowleaf Plantain.

Species in the genus Plantago have several unique traits that have led to them being adapted as model plants in various fields of study. However, the lack of a genetic manipulation system prevents in-depth investigation of gene function, limiting the versatility of this genus as a model. Here, a transformation protocol is presented for Plantago lanceolata, the most commonly studied Plantago species. Using Agrobacterium tumefaciens-mediated transformation, 3 week-old roots of aseptically grown P. lanceolata plants were infected with bacteria, incubated for 2-3 days, and then transferred to a shoot induction medium with appropriate antibiotic selection. Shoots typically emerged from the medium after 1 month, and roots developed 1-4 weeks after the shoots were transferred to the root induction medium. The plants were then acclimated to a soil environment and tested for the presence of a transgene using the β-glucuronidase (GUS) reporter assay. The transformation efficiency of the current method is ~20%, with two transgenic plants emerging per 10 root tissues transformed. Establishing a transformation protocol for narrowleaf plantain will facilitate the adoption of this plant as a new model species in various areas.

Genetic manipulation and targeted protein degradation in mammalian systems: practical considerations, tips and tricks for discovery research.

Gaining a mechanistic understanding of the molecular pathways underpinning cellular and organismal physiology invariably relies on the perturbation of an experimental system to infer causality. This can be achieved either by genetic manipulation or by pharmacological treatment. Generally, the former approach is applicable to a wider range of targets, is more precise, and can address more nuanced functional aspects. Despite such apparent advantages, genetic manipulation (i.e., knock-down, knock-out, mutation, and tagging) in mammalian systems can be challenging due to problems with delivery, low rates of homologous recombination, and epigenetic silencing. The advent of CRISPR-Cas9 in combination with the development of robust differentiation protocols that can efficiently generate a variety of different cell types in vitro has accelerated our ability to probe gene function in a more physiological setting. Often, the main obstacle in this path of enquiry is to achieve the desired genetic modification. In this short review, we will focus on gene perturbation in mammalian cells and how editing and differentiation of pluripotent stem cells can complement more traditional approaches. Additionally, we introduce novel targeted protein degradation approaches as an alternative to DNA/RNA-based manipulation. Our aim is to present a broad overview of recent approaches and in vitro systems to study mammalian cell biology. Due to space limitations, we limit ourselves to providing the inexperienced reader with a conceptual framework on how to use these tools, and for more in-depth information, we will provide specific references throughout.

From multiallele fish to nonstandard environments, how ZFIN assigns phenotypes, human disease models, and gene expression annotations to genes.

Danio rerio is a model organism used to investigate vertebrate development. Manipulation of the zebrafish genome and resultant gene products by mutation or targeted knockdown has made the zebrafish a good system for investigating gene function, providing a resource to investigate genetic contributors to phenotype and human disease. Phenotypic outcomes can be the result of gene mutation, targeted knockdown of gene products, manipulation of experimental conditions, or any combination thereof. Zebrafish have been used in various genetic and chemical screens to identify genetic and environmental contributors to phenotype and disease outcomes. The Zebrafish Information Network (ZFIN, zfin.org) is the central repository for genetic, genomic, and phenotypic data that result from research using D. rerio. Here we describe how ZFIN annotates phenotype, expression, and disease model data across various experimental designs, how we computationally determine wild-type gene expression, the phenotypic gene, and how these results allow us to propagate gene expression, phenotype, and disease model data to the correct gene, or gene related entity.

High-quality haplotype-resolved genome assembly of cultivated octoploid strawberry.

Cultivated strawberry (Fragaria × ananassa), a perennial herb belonging to the family Rosaceae, is a complex octoploid with high heterozygosity at most loci. However, there is no research on the haplotype of the octoploid strawberry genome. Here we aimed to obtain a high-quality genome of the cultivated strawberry cultivar, "Yanli", using single molecule real-time sequencing and high-throughput chromosome conformation capture technology. The "Yanli" genome was 823Mb in size, with a long terminal repeat assembly index of 14.99. The genome was phased into two haplotypes, Hap1 (825Mb with contig N50 of 26.70Mb) and Hap2 (808Mb with contig N50 of 27.51Mb). Using the combination of Hap1 and Hap2, we obtained for the first time a haplotype-resolved genome with 56 chromosomes for the cultivated octoploid strawberry. We identified a ~ 10Mb inversion and translocation on chromosome 2-1. 104 957 and 102 356 protein-coding genes were annotated in Hap1 and Hap2, respectively. Analysis of the genes related to the anthocyanin biosynthesis pathway revealed the structural diversity and complexity in the expression of the alleles in the octoploid F. × ananassa genome. In summary, we obtained a high-quality haplotype-resolved genome assembly of F. × ananassa, which will provide the foundation for investigating gene function and evolution of the genome of cultivated octoploid strawberry.

Establishing an Efficient Electroporation-Based Method to Manipulate Target Gene Expression in the Axolotl Brain

The tetrapod salamander species axolotl (Ambystoma mexicanum) is capable of regenerating injured brain. For better understanding the mechanisms of brain regeneration, it is very necessary to establish a rapid and efficient gain-of-function and loss-of-function approaches to study gene function in the axolotl brain. Here, we establish and optimize an electroporation-based method to overexpress or knockout/knockdown target gene in ependymal glial cells (EGCs) in the axolotl telencephalon. By orientating the electrodes, we were able to achieve specific expression of EGFP in EGCs located in dorsal, ventral, medial, or lateral ventricular zones. We then studied the role of Cdc42 in brain regeneration by introducing Cdc42 into EGCs through electroporation, followed by brain injury. Our findings showed that overexpression of Cdc42 in EGCs did not significantly affect EGC proliferation and production of newly born neurons, but it disrupted their apical polarity, as indicated by the loss of the ZO-1 tight junction marker. This disruption led to a ventricular accumulation of newly born neurons, which are failed to migrate into the neuronal layer where they could mature, thus resulted in a delayed brain regeneration phenotype. Furthermore, when electroporating CAS9-gRNA protein complexes against TnC (Tenascin-C) into EGCs of the brain, we achieved an efficient knockdown of TnC. In the electroporation-targeted area, TnC expression is dramatically reduced at both mRNA and protein levels. Overall, this study established a rapid and efficient electroporation-based gene manipulation approach allowing for investigation of gene function in the process of axolotl brain regeneration.

<i>Agrobacterium</i>-mediated transformation efficiency and grain phenotypes in six <i>indica</i> and <i>japonica</i> rice cultivars

Plant transformation and regeneration have been continuously developed for the past four decades. In rice (<italic>Oryza sativa</italic> L.), <italic>Agrobacterium</italic>-mediated transformation has high efficiency in<italic> japonica</italic> and some <italic>indica</italic> cultivars using mature seeds and immature embryos. However, these protocols have low transformation efficiency in the latest <italic>indica</italic> cultivar that has been developed in South China since 2010. Here, we explored plant culture regeneration of the high-quality and high-yield <italic>indica</italic> cultivar Nanguizhan (NGZ) through CRISPR/Cas-based genome editing and traditional overexpression transformations. We compared transformation efficiency in this cultivar to four other widely-used <italic>indica</italic> cultivars and one <italic>japonica</italic> cultivar using mature seeds and the gene <italic>Grain Size and Number 1</italic> (<italic>GSN1</italic>) as a case study. We observed universe smaller grain size in overexpression lines and bigger grain size in gene-editing lines among different cultivars. NGZ phenotypes make it an excellent model in which to investigate gene functions. We also examined the single nucleotide polymorphism (SNP) distribution and differences in expression levels of regeneration-related genes in calli, possibly revealing the source of NGZ's advantages in <italic>Agrobacterium</italic>-mediated transformation. These results shed light on the advanced application of NGZ in gene editing and overexpression transformation related to grain improvement, contributing to the "rice breeding 4.0 era".

An efficient marker-free genome editing method for Aspergillus niger

Aspergillus niger is an important industrial strain which has been widely used for production of enzymes and organic acids. Genome modification of A. niger is required to further improve its potential for industrial production. CRISPR/Cas9 is a widely used genome editing technique for A. niger, but its application in industrial strains modification is hampered by the need for integration of a selection marker into the genome or low gene editing efficiency. Here we report a highly efficient marker-free genome editing method for A. niger based on CRISPR/Cas9 technique. Firstly, we constructed a co-expression plasmid of sgRNA and Cas9 with a replication initiation region fragment AMA1 (autonomously maintained in Aspergillus) by using 5S rRNA promoter which improved sgRNA expression. Meanwhile, a strain deficient in non-homologous end-joining (NHEJ) was developed by knocking out the kusA gene. Finally, we took advantage of the instability of plasmid containing AMA1 fragment to cure the co-expression plasmid containing sgRNA and Cas9 through passaging on non-selective plate. With this method, the efficiency of gene editing reached 100% when using maker-free donor DNA with a short homologous arm of 20 bp. This method may facilitate investigation of gene functions and construction of cell factories for A. niger.

Regulation of pre-mRNA splicing: roles in physiology and disease, and therapeutic prospects.

The removal of introns from mRNA precursors and its regulation by alternative splicing are key for eukaryotic gene expression and cellular function, as evidenced by the numerous pathologies induced or modified by splicing alterations. Major recent advances have been made in understanding the structures and functions of the splicing machinery, in the description and classification of physiological and pathological isoforms and in the development of the first therapies for genetic diseases based on modulation of splicing. Here, we review this progress and discuss important remaining challenges, including predicting splice sites from genomic sequences, understanding the variety of molecular mechanisms and logic of splicing regulation, and harnessing this knowledge for probing gene function and disease aetiology and for the design of novel therapeutic approaches.

Simultaneous loading of PCR-based multiple fragments on mouse artificial chromosome vectors in DT40 cell for gene delivery

Homology-directed repair-mediated knock-in (HDR-KI) in combination with CRISPR-Cas9-mediated double strand break (DSB) leads to high frequency of site-specific HDR-KI. While this characteristic is advantageous for generating genetically modified cellular and animal models, HDR-KI efficiency in mammalian cells remains low. Since avian DT40 cells offer distinct advantage of high HDR-KI efficiency, we expanded this practicality to adapt to mammalian research through sequential insertion of target sequences into mouse/human artificial chromosome vector (MAC/HAC). Here, we developed the simultaneous insertion of multiple fragments by HDR method termed the simHDR wherein a target sequence and selection markers could be loaded onto MAC simultaneously. Additionally, preparing each HDR donor containing homology arm by PCR could bypass the cloning steps of target sequence and selection markers. To confirm the functionality of the loaded HDR donors, we constructed a MAC with human leukocyte antigen A (HLA-A) gene in the DT40 cells, and verified the expression of this genomic region by reverse transcription PCR (RT-PCR) and western blotting. Collectively, the simHDR offers a rapid and convenient approach to generate genetically modified models for investigating gene functions, as well as understanding disease mechanisms and therapeutic interventions.

Workflow for Performing Genetic Manipulation in Human Trophoblast Stem Cells Using CRISPR/Cas9 Technology.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based tool kits greatly facilitate the investigation of gene function, especially gene editing and transcriptional regulation technology. Human trophoblast stem cell (hTSC) is a promising cell model for simulating the development and function of human placental trophoblast cells. Here, we provide an optimized workflow for efficient genome editing and transcriptional activation in hTSCs using a lentivirus-dependent method. We also present methods to evaluate the gene targeted mutations and transcriptional activation.

Prime editing in chicken fibroblasts and primordial germ cells.

CRISPR/Cas9-based genome editing technologies are revolutionizing developmental biology. One of the advanced CRISPR-based techniques is prime editing (PE), which enables precise gene modification in multiple model organisms. However, there has been no report of taking advantage of the PE system for gene editing in primordial germ cells (PGCs) thus far. In the current study, we describe a method to apply PE to the genome of chicken fibroblasts and PGCs. By combining PE with a transposon-mediated genomic integration, drug selection, and the single-cell culture method, we successfully generated prime-edited chicken fibroblasts and PGCs. The chicken PGC is widely used as an experimental model to study germ cell formation and as a vector for gene transfer to produce transgenic chickens. Such experimental models are useful in the developmental biology field and as potential bioreactors to produce pharmaceutical and nutritious proteins. Thus, the method presented here will provide not only a powerful tool to investigate gene function in germ cell development but also a basis for generating prime-edited transgenic birds.

Small Interfering RNA Delivery Into Primary Human Natural Killer Cells for Functional Gene Analyses

Studying gene functions in human natural killer (NK) cells is key for advancing the understanding of NK cell biology and holds promise to pave the way for improving NK cell therapies against cancer. However, NK cells are challenging to manipulate, and investigation of gene functions in NK cells is hampered by variable delivery efficiencies and impaired viability upon electroporation, lipofection, or viral transduction. Here, we report a simple workflow for delivery of commercially available small interfering RNA molecules into primary human NK cells to enable functional gene analyses. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Enrichment of natural killer cells from human peripheral blood mononuclear cells Basic Protocol 2: Preparation of small interfering RNA Basic Protocol 3: Delivery of small interfering RNA into natural killer cells Support Protocol 1: Isolation of human peripheral blood mononuclear cells from buffy coats Support Protocol 2: Thawing and recovery of cryopreserved peripheral blood mononuclear cells Support Protocol 3: Evaluation of natural killer cell purity following magnetic enrichment Support Protocol 4: Exemplary assessment of knockdown efficiency.

Discovery of a MUC3B gene reconstructs the membrane mucin gene cluster on human chromosome 7.

Human tissue surfaces are coated with mucins, a family of macromolecular sugar-laden proteins serving diverse functions from lubrication to the formation of selective biochemical barriers against harmful microorganisms and molecules. Membrane mucins are a distinct group of mucins that are attached to epithelial cell surfaces where they create a dense glycocalyx facing the extracellular environment. All mucin proteins carry long stretches of tandemly repeated sequences that undergo extensive O-linked glycosylation to form linear mucin domains. However, the repetitive nature of mucin domains makes them prone to recombination and renders their genetic sequences particularly difficult to read with standard sequencing technologies. As a result, human mucin genes suffer from significant sequence gaps that have hampered the investigation of gene function in health and disease. Here we leveraged a recent human genome assembly to characterize a previously unmapped MUC3B gene located at the q22 locus on chromosome 7, within a cluster of four structurally related membrane mucin genes that we name the MUC3 cluster. We found that MUC3B shares high sequence identity with the known MUC3A gene and that the two genes are governed by evolutionarily conserved regulatory elements. Furthermore, we show that MUC3A, MUC3B, MUC12, and MUC17 in the human MUC3 cluster are expressed in intestinal epithelial cells (IECs). Our results complete existing genetic gaps in the MUC3 cluster which is a conserved genetic unit in vertebrates. We anticipate our results to be the starting point for the detection of disease-associated polymorphisms in the human MUC3 cluster. Moreover, our study provides the basis for the exploration of intestinal mucin gene function in widely used experimental models such as human intestinal organoids and genetic mouse models.

CEDA: integrating gene expression data with CRISPR-pooled screen data identifies essential genes with higher expression.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based genetic perturbation screen is a powerful tool to probe gene function. However, experimental noises, especially for the lowly expressed genes, need to be accounted for to maintain proper control of false positive rate. We develop a statistical method, named CRISPR screen with Expression Data Analysis (CEDA), to integrate gene expression profiles and CRISPR screen data for identifying essential genes. CEDA stratifies genes based on expression level and adopts a three-component mixture model for the log-fold change of single-guide RNAs (sgRNAs). Empirical Bayesian prior and expectation-maximization algorithm are used for parameter estimation and false discovery rate inference. Taking advantage of gene expression data, CEDA identifies essential genes with higher expression. Compared to existing methods, CEDA shows comparable reliability but higher sensitivity in detecting essential genes with moderate sgRNA fold change. Therefore, using the same CRISPR data, CEDA generates an additional hit gene list. Supplementary data are available at Bioinformatics online.

A Comparison of RNA Interference via Injection and Feeding in Honey Bees.

Simple SummaryRNA interference is an important way to analyze gene function. It is also widely used in honey bees. It was thought that RNA interference with honey bee brain genes could only be achieved by injection. However, this method of injection is very complicated and can easily cause damage to bees. Recent studies have shown that specially treated siRNA can knockdown brain genes in animals by feeding them. Therefore, we used different approaches to deliver modified and unmodified siRNAs to investigate what methods can successfully interfere with honey bee brain genes. The results of this study are helpful to improve the application of RNA interference techniques in honey bees and other insects.RNA interference (RNAi) has been used successfully to reduce target gene expression and induce specific phenotypes in several species. It has proved useful as a tool to investigate gene function and has the potential to manage pest populations and reduce disease pathogens. However, it is not known whether different administration methods are equally effective at interfering with genes in bees. Therefore, we compared the effects of feeding and injection of small interfering RNA (siRNA) on the messenger RNA (mRNA) levels of alpha-aminoadipic semialdehyde dehydrogenase (ALDH7A1), 4-coumarate-CoA ligase (4CL), and heat shock protein 70 (HSP70). Both feeding and injection of siRNA successfully knocked down the gene but feeding required more siRNA than the injection. Our results suggest that both feeding and injection of siRNA effectively interfere with brain genes in bees. The appropriateness of each method would depend on the situation.

In-depth analysis of genomes and functional genomics of orchid using cutting-edge high-throughput sequencing.

High-throughput sequencing technology has been facilitated the development of new methodologies and approaches for studying the origin and evolution of plant genomes and subgenomes, population domestication, and functional genomics. Orchids have tens of thousands of members in nature. Many of them have promising application potential in the extension and conservation of the ecological chain, the horticultural use of ornamental blossoms, and the utilization of botanical medicines. However, a large-scale gene knockout mutant library and a sophisticated genetic transformation system are still lacking in the improvement of orchid germplasm resources. New gene editing tools, such as the favored CRISPR-Cas9 or some base editors, have not yet been widely applied in orchids. In addition to a large variety of orchid cultivars, the high-precision, high-throughput genome sequencing technology is also required for the mining of trait-related functional genes. Nowadays, the focus of orchid genomics research has been directed to the origin and classification of species, genome evolution and deletion, gene duplication and chromosomal polyploidy, and flower morphogenesis-related regulation. Here, the progressing achieved in orchid molecular biology and genomics over the past few decades have been discussed, including the evolution of genome size and polyploidization. The frequent incorporation of LTR retrotransposons play important role in the expansion and structural variation of the orchid genome. The large-scale gene duplication event of the nuclear genome generated plenty of recently tandem duplicated genes, which drove the evolution and functional divergency of new genes. The evolution and loss of the plastid genome, which mostly affected genes related to photosynthesis and autotrophy, demonstrated that orchids have experienced more separate transitions to heterotrophy than any other terrestrial plant. Moreover, large-scale resequencing provide useful SNP markers for constructing genetic maps, which will facilitate the breeding of novel orchid varieties. The significance of high-throughput sequencing and gene editing technologies in the identification and molecular breeding of the trait-related genes in orchids provides us with a representative trait-improving gene as well as some mechanisms worthy of further investigation. In addition, gene editing has promise for the improvement of orchid genetic transformation and the investigation of gene function. This knowledge may provide a scientific reference and theoretical basis for orchid genome studies.

Identification of key genes involved in calcific aortic valve disease based on integrated bioinformatics analysis.

The calcific aortic valve disease (CAVD) develops as an aortic valve sclerosis and progresses to an advanced form of stenosis. In many biological fields, bioinformatics becomes a fundamental component. The key mechanisms involved in CAVD are discovered with the use of bioinformatics to investigate gene function and pathways. We downloaded the original data (GSE51472) from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/). After standardization, 2978 differentially expressed genes (DEGs) were identified from the data sets GSE51472 containing samples from normal, calcified, and sclerotic aortic valves. Analysis of DEGs based on the series test of clusters (STCs) revealed the two most significant patterns. Based on the result of the STC, the functional enrichment analysis of gene ontology (GO) was conducted to investigate the molecular function (MF), biological process (BP), and cell compound (CC) of the DEGs. With a p value of 0.01, DEGs associated with "chronic inflammation," "T-cell receptor complexes," and "antigen binding" had the highest significance within BP, CC, and MF. DEG enrichment in signaling pathways was analyzed using KEGG pathway enrichment. Using a p < 0.05 level of significance, the most enriched biological pathways related to CAVD were "Chemokine signaling pathway," "Cytokine-cytokine receptor interaction," "Tuberculosis," "PI3K-Akt signaling pathway," and "Transcriptional misregulation in cancer." Finally, the construction of gene co-expression networks and pathway networks illustrated the pathogensis of CAVD. TLR2, CD86, and TYROBP were identified as hub genes for the development of CAVD. Moreover, "MAPK signaling pathway," "Apoptosis," and "Pathways in cancer" were regarded as the core pathways among the samples of normal, sclerotic and calcified aortic valve samples.

Cre toxicity in mouse models of cardiovascular physiology and disease.

The Cre-LoxP system provides a widely used method for studying gene requirements in the mouse as the main mammalian genetic model organism. To define the molecular and cellular mechanisms that underlie cardiovascular development, function and disease, various mouse strains have been engineered that allow Cre-LoxP-mediated gene targeting within specific cell types of the cardiovascular system. Despite the usefulness of this system, evidence is accumulating that Cre activity can have toxic effects in cells, independently of its ability to recombine pairs of engineered LoxP sites in target genes. Here, we have gathered published evidence for Cre toxicity in cells and tissues relevant to cardiovascular biology and provide an overview of mechanisms proposed to underlie Cre toxicity. Based on this knowledge, we propose that each study utilising the Cre-LoxP system to investigate gene function in the cardiovascular system should incorporate appropriate controls to account for Cre toxicity.

Genome-Wide Association Study of Obstructive Sleep Apnea and Objective Sleep-related Traits Identifies Novel Risk Loci in Han Chinese Individuals.

Rationale: Previous genetic studies of obstructive sleep apnea (OSA) have limitations in terms of precise case definition, integrated quantitative traits, and interpretation of genetic functions; thus, the heritability of OSA remains poorly explained. Objectives: To identify novel genetic variants associated with OSA and objective sleep-related traits and to explore their functional roles. Methods: A genome-wide association study was performed in 20,590 Han Chinese individuals (5,438 OSA and 15,152 control samples). Human samples and point mutation knockin mice were used for follow-up investigation of gene functions. Measurements and Main Results: Two characteristic study-wide significant loci (P < 2.63 × 10-9) for OSA were identified: the PACRG intronic variant rs6455893 on 6q26 (odds ratio [OR] = 1.62; 95% confidence interval [CI], 1.39-1.89; P = 6.98 × 10-10) and the missense variant rs3746804 (p.Pro267Leu) in the riboflavin transporter SLC52A3 on 20p13 (OR = 0.83; 95% CI, 0.79-0.88; P = 7.57 × 10-10). In addition, 18 genome-wide significant loci associated with quantitative OSA and objective sleep-related traits were identified, 5 of which exceeded the study-wide significance threshold. Rs3746804 was associated with elevated serum riboflavin concentrations, and the corresponding mutation in mice increased riboflavin concentrations, suggesting that this variant may facilitate riboflavin uptake and riboflavin-dependent physiological activity. Conclusions: We identified several novel genome-wide significant loci associated with OSA and objective sleep-related traits. Our findings provide insight into the genetic architecture of OSA and suggest that SLC52A3 might be a therapeutic target, whereas riboflavin might be a therapeutic agent.