Corrigendum: Development of a microneedle swab for acquisition of genomic DNA from buccal cells.

[This corrects the article DOI: 10.3389/fbioe.2022.829648.].

Development of a Microneedle Swab for Acquisition of Genomic DNA From Buccal Cells.

A swab is a tool for obtaining buccal DNA from buccal mucus for biological analysis. The acquisition of a sufficient amount and high quality of DNA is an important factor in determining the accuracy of a diagnosis. A microneedle swab (MN swab) was developed to obtain more oral mucosal tissues non-invasively. Eight types of MN swabs were prepared with varying combinations of patterns (zigzag or straight), number of MNs, intervals of MNs, and sharpness of tips. When MN swab was applied up to 10 times, the tissue amount and DNA yield increased compared to commercial swabs. A zigzag pattern of microneedles was found to be more efficient than a straight pattern and increasing the number of microneedles in an array increased the DNA yield. The MN swab collected about twice the DNA compared to the commercial swab. In an in vivo test using mini pigs, the lower cycle threshold values of mucosal samples collected with MN swabs compared to samples collected with commercial swabs indicated that a greater amount of DNA was collected for SNP genotyping. A polymer MN swab is easy to manufacture by a single molding process, and it has a greater sampling capacity than existing commercial swabs.

Programmable Nuclease-Based Integration into Novel Extragenic Genomic Safe Harbor Identified from Korean Population-Based CNV Analysis.

Here, we found two genomic safe harbor (GSH) candidates from chromosomes 3 and 8, based on large-scale population-based cohort data from 4,694 Koreans by CNV analysis. Furthermore, estimated genotype of these CNVRs was validated by quantitative real-time PCR, and epidemiological data examined no significant genetic association between diseases or traits and two CNVRs. After screening the GSH candidates by in silico approaches, we designed TALEN pairs to integrate EGFP expression cassette into human cell lines in order to confirm the functionality of GSH candidates in an in vitro setting. As a result, transgene insertion into one of the two loci using TALEN showed robust transgene expression comparable to that with an AAVS1 site without significantly perturbing neighboring genes. Changing the promoter or cell type did not noticeably disturb this trend. Thus, we could validate two CNVRs as a site for effective and safe transgene insertion in human cells.

Exome chip-driven association study of lipidemia in >14,000 Koreans and evaluation of genetic effect on identified variants between different ethnic groups.

Lipid levels in blood are widely used to diagnose and monitor chronic diseases. It is essential to identify the genetic traits involved in lipid metabolism for understanding chronic diseases. However, the influence of genetic traits varies depending on race, sex, age, and ethnicity. Therefore, research focusing on populations of individual countries is required, and the results can be used as a basis for comparison of results of other studies at the cross-racial and cross-country levels. In the present study, we selected lipid-related variants and evaluated their effects on lipid-related diseases in more than 14,000 subjects of three cohorts using the Illumina Human Exome Beadchip. A genome-wide association study was conducted using EPACTs after adjusting for age, sex, and recruitment area. A genome-wide significance cutoff was defined as p < 5E-08 in all the three cohorts. Sixteen variants represented the lipid traits and were classified as vulnerable to borderline hypertriglyceridemia, hyper-LDL-cholesterolemia, or hypo-HDL-cholesterolemia. Moreover, we compared the genetic effects of the 16 variants between ethnic groups and identified the missense variants in apolipoprotein A-V, cholesterol ester transfer protein, and apolipoprotein E as Asian-specific. Our study provides candidate genes as markers for chronic diseases through the evaluation of genetic effects.

Dual Stimuli-Triggered Nanogels in Response to Temperature and pH Changes for Controlled Drug Release.

Poly-N-isopropyl acrylamide (PNIPAM) nanogels have been modified with different acrylic acid (AAc) contents for the efficient control of lower critical solution temperature (LCST). In this study, PNIPAM-co-AAc nanogels nanogels showed two volume phase transitions in comparison with PNIPAM. The transition temperature of PNIPAM nanogels was increased with AAc contents. The controlled drug release performance of PNIPAM-co-AAc nanogels loaded with ß-lapachone was attributed to the AAc content ratio and was efficiently triggered in response to temperature and pH. Moreover, a colorimetric cell proliferation assay and direct fluorescence-based live/dead staining were used to confirm the concurrence on drug release profiles. Finally, PNIPAM-co-AAc20 showed a relatively low level of drug release in the range of acidic to neutral pH at body temperature, while maximizing drug release at basic pH. Therefore, we demonstrated that the PNIPAM-based nanogel with the temperature- and pH-responsive features could be a promising nanocarrier for potential intestine-specific drug delivery.

The Korea Biobank Array: Design and Identification of Coding Variants Associated with Blood Biochemical Traits.

We introduce the design and implementation of a new array, the Korea Biobank Array (referred to as KoreanChip), optimized for the Korean population and demonstrate findings from GWAS of blood biochemical traits. KoreanChip comprised >833,000 markers including >247,000 rare-frequency or functional variants estimated from >2,500 sequencing data in Koreans. Of the 833 K markers, 208 K functional markers were directly genotyped. Particularly, >89 K markers were presented in East Asians. KoreanChip achieved higher imputation performance owing to the excellent genomic coverage of 95.38% for common and 73.65% for low-frequency variants. From GWAS (Genome-wide association study) using 6,949 individuals, 28 associations were successfully recapitulated. Moreover, 9 missense variants were newly identified, of which we identified new associations between a common population-specific missense variant, rs671 (p.Glu457Lys) of ALDH2, and two traits including aspartate aminotransferase (P = 5.20 × 10-13) and alanine aminotransferase (P = 4.98 × 10-8). Furthermore, two novel missense variants of GPT with rare frequency in East Asians but extreme rarity in other populations were associated with alanine aminotransferase (rs200088103; p.Arg133Trp, P = 2.02 × 10-9 and rs748547625; p.Arg143Cys, P = 1.41 × 10-6). These variants were successfully replicated in 6,000 individuals (P = 5.30 × 10-8 and P = 1.24 × 10-6). GWAS results suggest the promising utility of KoreanChip with a substantial number of damaging variants to identify new population-specific disease-associated rare/functional variants.

Genome-Wide Association Study Meta-Analysis of Long-Term Average Blood Pressure in East Asians.

BACKGROUND: Genome-wide single marker and gene-based meta-analyses of long-term average (LTA) blood pressure (BP) phenotypes may reveal novel findings for BP. METHODS AND RESULTS: We conducted genome-wide analysis among 18 422 East Asian participants (stage 1) followed by replication study of ≤46 629 participants of European ancestry (stage 2). Significant single-nucleotide polymorphisms and genes were determined by a P<5.0×10-8 and 2.5×10-6, respectively, in joint analyses of stage-1 and stage-2 data. We identified 1 novel ARL3 variant, rs4919669 at 10q24.32, influencing LTA systolic BP (stage-1 P=5.03×10-8, stage-2 P=8.64×10-3, joint P=2.63×10-8) and mean arterial pressure (stage-1 P=3.59×10-9, stage-2 P=2.35×10-2, joint P=2.64×10-8). Three previously reported BP loci (WBP1L, NT5C2, and ATP2B1) were also identified for all BP phenotypes. Gene-based analysis provided the first robust evidence for association of KCNJ11 with LTA systolic BP (stage-1 P=8.55×10-6, stage-2 P=1.62×10-5, joint P=3.28×10-9) and mean arterial pressure (stage-1 P=9.19×10-7, stage-2 P=9.69×10-5, joint P=2.15×10-9) phenotypes. Fourteen genes (TMEM180, ACTR1A, SUFU, ARL3, SFXN2, WBP1L, CYP17A1, C10orf32, C10orf32-ASMT, AS3MT, CNNM2, and NT5C2 at 10q24.32; ATP2B1 at 12q21.33; and NCR3LG1 at 11p15.1) implicated by previous genome-wide association study meta-analyses were also identified. Among the loci identified by the previous genome-wide association study meta-analysis of LTA BP, we transethnically replicated associations of the KCNK3 marker rs1275988 at 2p23.3 with LTA systolic BP and mean arterial pressure phenotypes (P=1.27×10-4 and 3.30×10-4, respectively). CONCLUSIONS: We identified 1 novel variant and 1 novel gene and present the first direct evidence of relevance of the KCNK3 locus for LTA BP among East Asians.

Genome-based exome sequencing analysis identifies GYG1, DIS3L and DDRGK1 are associated with myocardial infarction in Koreans.

Myocardial infarction (MI) is a complex disease caused by combination of genetic and environmental factors. Although genome-wide association studies (GWAS) identified more than 46 risk loci which are associated with coronary artery disease and MI, most of the genetic variability inMI still remains undefined. Here, we screened the susceptibility loci for MI using exome sequencing and validated candidate variants in replication sets. We identified that three genes (GYG1, DIS3L and DDRGK1) were associated with MI at the discovery and replication stages. Further research will be required to determine the functional association of these genes with MI risk, and these associations have to be confirmed in other ethnic populations.

Association of Metabolites with Obesity and Type 2 Diabetes Based on FTO Genotype.

The single nucleotide polymorphism rs9939609 of the gene FTO, which encodes fat mass and obesity-associated protein, is strongly associated with obesity and type 2 diabetes (T2D) in multiple populations; however, the underlying mechanism of this association is unclear. The present study aimed to investigate FTO genotype-dependent metabolic changes in obesity and T2D. To elucidate metabolic dysregulation associated with disease risk genotype, genomic and metabolomic datasets were recruited from 2,577 participants of the Korean Association REsource (KARE) cohort, including 40 homozygous carriers of the FTO risk allele (AA), 570 heterozygous carriers (AT), and 1,967 participants carrying no risk allele (TT). A total of 134 serum metabolites were quantified using a targeted metabolomics approach. Through comparison of various statistical methods, seven metabolites were identified that are significantly altered in obesity and T2D based on the FTO risk allele (adjusted p < 0.05). These identified metabolites are relevant to phosphatidylcholine metabolic pathway, and previously reported to be metabolic markers of obesity and T2D. In conclusion, using metabolomics with the information from genome-wide association studies revealed significantly altered metabolites depending on the FTO genotype in complex disorders. This study may contribute to a better understanding of the biological mechanisms linking obesity and T2D.

Evaluation of pleiotropic effects among common genetic loci identified for cardio-metabolic traits in a Korean population.

BACKGROUND: The genetic contribution to complex diseases or traits, including cardio-metabolic traits, has been elucidated recently by large-scale genome-wide association studies. These genome-wide association studies have indicated that most pleiotropic loci contain genes associated with lipids. Clinically, lipid related abnormalities are strongly associated with other diseases such as type 2 diabetes, coronary artery disease and hypertension. The aim of this study was to evaluate the shared genetic background of lipids and other cardio-metabolic traits. METHODS: We conducted meta-analyses of the association between 157 published lipid-associated loci and 10 cardio-metabolic traits in 14,028 Korean individuals genotyped using the Exome chip (Illumina HumanExome BeadChip). We also examined whether the pleiotropic effects of such loci constituted independent (i.e., biological) pleiotropy or mediated pleiotropy in these metabolic pathways. RESULTS: Eighteen lipid-associated loci were significantly associated with one of six cardio-metabolic traits after correction for multiple testing (P < 3.70 × 10(-4)). Region 12q24.12 had pleiotropic effects on fasting plasma glucose, blood pressure and obesity-related traits (body mass index and waist-hip ratio) independent of its effects on the lipid profile. Lipid risk scores, calculated according to whether or not subjects carried the risk allele for lipid traits, were significantly associated with fasting plasma glucose, blood pressure and obesity-related traits. CONCLUSIONS: The 12q24.12 region showed ethnic-specific genetic pleiotropy among cardio-metabolic traits in this study. Our findings may help to account for molecular mechanisms based on shared genetic background underlying not only dyslipidemia, but also cardiovascular disease and type 2 diabetes.

A comprehensive 1,000 Genomes-based genome-wide association meta-analysis of coronary artery disease.

Existing knowledge of genetic variants affecting risk of coronary artery disease (CAD) is largely based on genome-wide association study (GWAS) analysis of common SNPs. Leveraging phased haplotypes from the 1000 Genomes Project, we report a GWAS meta-analysis of ∼185,000 CAD cases and controls, interrogating 6.7 million common (minor allele frequency (MAF) > 0.05) and 2.7 million low-frequency (0.005 < MAF < 0.05) variants. In addition to confirming most known CAD-associated loci, we identified ten new loci (eight additive and two recessive) that contain candidate causal genes newly implicating biological processes in vessel walls. We observed intralocus allelic heterogeneity but little evidence of low-frequency variants with larger effects and no evidence of synthetic association. Our analysis provides a comprehensive survey of the fine genetic architecture of CAD, showing that genetic susceptibility to this common disease is largely determined by common SNPs of small effect size.

Trans-ancestry genome-wide association study identifies 12 genetic loci influencing blood pressure and implicates a role for DNA methylation.

We carried out a trans-ancestry genome-wide association and replication study of blood pressure phenotypes among up to 320,251 individuals of East Asian, European and South Asian ancestry. We find genetic variants at 12 new loci to be associated with blood pressure (P = 3.9 × 10(-11) to 5.0 × 10(-21)). The sentinel blood pressure SNPs are enriched for association with DNA methylation at multiple nearby CpG sites, suggesting that, at some of the loci identified, DNA methylation may lie on the regulatory pathway linking sequence variation to blood pressure. The sentinel SNPs at the 12 new loci point to genes involved in vascular smooth muscle (IGFBP3, KCNK3, PDE3A and PRDM6) and renal (ARHGAP24, OSR1, SLC22A7 and TBX2) function. The new and known genetic variants predict increased left ventricular mass, circulating levels of NT-proBNP, and cardiovascular and all-cause mortality (P = 0.04 to 8.6 × 10(-6)). Our results provide new evidence for the role of DNA methylation in blood pressure regulation.

Influence of Genetic Variants in EGF and Other Genes on Hematological Traits in Korean Populations by a Genome-Wide Approach.

Hematological traits are important health indicators and are used as diagnostic clinical parameters for human disorders. Recently, genome-wide association studies (GWAS) identified many genetic loci associated with hematological traits in diverse ethnic groups. However, additional GWAS are necessary to elucidate the breadth of genetic variation and the underlying genetic architecture represented by hematological metrics. To identify additional genetic loci influencing hematological traits (such as hematocrit, hemoglobin concentration, white blood cell count, red blood cell count, and platelet count), we conducted GWAS and meta-analyses on data from 12,509 Korean individuals grouped into population-based cohorts. Of interest is EGF, a factor plays a role in the proliferation and differentiation of hematopoietic progenitor cells. We identified a novel EGF variant, which associated with platelet count in our study (P combined = 2.44 × 10(-15)). Our study also replicated 16 genetic associations related to five hematological traits with genome-wide significance (P < 5 × 10(-8)) that were previously established in other ethnic groups. Of these, variants influencing platelet count are distributed across several genes and have pleiotropic effects in coronary artery disease and dyslipidemia. Our findings may aid in elucidating molecular mechanisms underlying not only hematopoiesis but also inflammatory and cardiovascular diseases.

Combinatorial approach to estimate copy number genotype using whole-exome sequencing data.

Copy number variations (CNVs) are known risk factors in complex diseases. Array-based approaches have been widely used to detect CNVs, but limitations of array-based CNV detection methods, such as noisy signal and low resolution, have hindered detection of small CNVs. Recently, the development of next-generation sequencing techniques has increased rapidly owing to declines in cost. Particularly, whole-exome sequencing has proved useful for finding causal genes and variants in complex diseases. Because gene copy number may affect expression, CNV genotyping can be very valuable in disease association studies. However, almost all current CNV detection tools consider only two types of CNV genotypes. In this study, we propose a CNV genotype estimation approach using a combination of existing methods. Our approach was comprehensively compared with the customized Agilent array-comparative genomic hybridization. We found that our genotyping approach proved to be accurate, and reproducible, suggesting that it can complement existing CNV genotyping methods.

Identification of a genetic variant at 2q12.1 associated with blood pressure in East Asians by genome-wide scan including gene-environment interactions.

BACKGROUND: Genome-wide association studies have identified many genetic loci associated with blood pressure (BP). Genetic effects on BP can be altered by environmental exposures via multiple biological pathways. Especially, obesity is one of important environmental risk factors that can have considerable effect on BP and it may interact with genetic factors. Given that, we aimed to test whether genetic factors and obesity may jointly influence BP. METHODS: We performed meta-analyses of genome-wide association data for systolic blood pressure (SBP) and diastolic blood pressure (DBP) that included analyses of interaction between single nucleotide polymorphisms (SNPs) and the obesity-related anthropometric measures, body mass index (BMI), height, weight, and waist/hip ratio (WHR) in East-Asians (n = 12,030). RESULTS: We identified that rs13390641 on 2q12.1 demonstrated significant association with SBP when the interaction between SNPs and BMI was considered (P < 5 × 10 -8). The gene located nearest to rs13390641, TMEM182, encodes transmembrane protein 182. In stratified analyses, the effect of rs13390641 on BP was much stronger in obese individuals (BMI ≥ 30) than non-obese individuals and the effect of BMI on BP was strongest in individuals with the homozygous A allele of rs13390641. CONCLUSIONS: Our analyses that included interactions between SNPs and environmental factors identified a genetic variant associated with BP that was overlooked in standard analyses in which only genetic factors were included. This result also revealed a potential mechanism that integrates genetic factors and obesity related traits in the development of high BP.

Evaluating the effects of CELF1 deficiency in a mouse model of RNA toxicity.

Myotonic dystrophy type 1 (DM1), the most common form of adult-onset muscular dystrophy, is caused by an expanded (CTG)n repeat in the 3' untranslated region of the DM protein kinase (DMPK) gene. The toxic RNA transcripts produced from the mutant allele alter the function of RNA-binding proteins leading to the functional depletion of muscleblind-like (MBNL) proteins and an increase in steady state levels of CUG-BP1 (CUGBP-ETR-3 like factor 1, CELF1). The role of increased CELF1 in DM1 pathogenesis is well studied using genetically engineered mouse models. Also, as a potential therapeutic strategy, the benefits of increasing MBNL1 expression have recently been reported. However, the effect of reduction of CELF1 is not yet clear. In this study, we generated CELF1 knockout mice, which also carry an inducible toxic RNA transgene to test the effects of CELF1 reduction in RNA toxicity. We found that the absence of CELF1 did not correct splicing defects. It did however mitigate the increase in translational targets of CELF1 (MEF2A and C/EBPß). Notably, we found that loss of CELF1 prevented deterioration of muscle function by the toxic RNA, and resulted in better muscle histopathology. These data suggest that while reduction of CELF1 may be of limited benefit with respect to DM1-associated spliceopathy, it may be beneficial to the muscular dystrophy associated with RNA toxicity.

Ribosomal protein S6, a target of rapamycin, is involved in the regulation of rRNA genes by possible epigenetic changes in Arabidopsis.

The target of rapamycin (TOR) kinase pathway regulates various biological processes, including translation, synthesis of ribosomal proteins, and transcription of rRNA. The ribosomal protein S6 (RPS6) is one of the well known downstream components of the TOR pathway. Ribosomal proteins have been known to have diverse functions in regulating cellular metabolism as well as protein synthesis. So far, however, little is known about other possible role(s) of RPS6 in plants, besides being a component of the 40 S ribosomal subunit and acting as a target of TOR. Here, we report that RPS6 may have a novel function via interaction with histone deacetylase 2B (AtHD2B) that belongs to the plant-specific histone deacetylase HD2 family. RPS6 and AtHD2B were localized to the nucleolus. Co-expression of RPS6 and AtHD2B caused a change in the location of both RPS6 and AtHD2B to one or several nucleolar spots. ChIP analysis suggests that RPS6 directly interacts with the rRNA gene promoter. Protoplasts overexpressing both AtHD2B and RPS6 exhibited down-regulation of pre-18 S rRNA synthesis with a concomitant decrease in transcription of some of the ribosomal proteins, suggesting their direct role in ribosome biogenesis and plant development. This is consistent with the mutation in rps6b that results in reduction in 18 S rRNA transcription and decreased root growth. We propose that the interaction between RPS6 and AtHD2B brings about a change in the chromatin structure of rDNA and thus plays an important role in linking TOR signaling to rDNA transcription and ribosome biogenesis in plants.

Genome-wide association study identifies candidate Loci associated with platelet count in koreans.

Platelets are derived from the fragments that are formed from the cytoplasm of bone marrow megakaryocytes-small irregularly shaped anuclear cells. Platelets respond to vascular damage, contracts blood vessels, and attaches to the damaged region, thereby stopping bleeding, together with the action of blood coagulation factors. Platelet activation is known to affect genes associated with vascular risk factors, as well as with arteriosclerosis and myocardial infarction. Here, we performed a genome-wide association study with 352,228 single-nucleotide polymorphisms typed in 8,842 subjects of the Korea Association Resource (KARE) project and replicated the results in 7,861 subjects from an independent population. We identified genetic associations between platelet count and common variants nearby chromosome 4p16.1 (p = 1.46 × 10(-10), in the KIAA0232 gene), 6p21 (p = 1.36 × 10(-7), in the BAK1 gene), and 12q24.12 (p = 1.11 × 10(-15), in the SH2B3 gene). Our results illustrate the value of large-scale discovery and a focus for several novel research avenues.

Functional implication of ß-carotene hydroxylases in soybean nodulation.

Legume-Rhizobium spp. symbiosis requires signaling between the symbiotic partners and differential expression of plant genes during nodule development. Previously, we cloned a gene encoding a putative ß-carotene hydroxylase (GmBCH1) from soybean (Glycine max) whose expression increased during nodulation with Bradyrhizobium japonicum. In this work, we extended our study to three GmBCHs to examine their possible role(s) in nodule development, as they were additionally identified as nodule specific, along with the completion of the soybean genome. In situ hybridization revealed the expression of three GmBCHs (GmBCH1, GmBCH2, and GmBCH3) in the infected cells of root nodules, and their enzymatic activities were confirmed by functional assays in Escherichia coli. Localization of GmBCHs by transfecting Arabidopsis (Arabidopsis thaliana) protoplasts with green fluorescent protein fusions and by electron microscopic immunogold detection in soybean nodules indicated that GmBCH2 and GmBCH3 were present in plastids, while GmBCH1 appeared to be cytosolic. RNA interference of the GmBCHs severely impaired nitrogen fixation as well as nodule development. Surprisingly, we failed to detect zeaxanthin, a product of GmBCH, or any other carotenoids in nodules. Therefore, we examined the possibility that most of the carotenoids in nodules are converted or cleaved to other compounds. We detected the expression of some carotenoid cleavage dioxygenases (GmCCDs) in wild-type nodules and also a reduced amount of zeaxanthin in GmCCD8-expressing E. coli, suggesting cleavage of the carotenoid. In view of these findings, we propose that carotenoids such as zeaxanthin synthesized in root nodules are cleaved by GmCCDs, and we discuss the possible roles of the carotenoid cleavage products in nodulation.

RNA interference-mediated repression of S6 kinase 1 impairs root nodule development in soybean.

Symbiotic nodule formation on legume roots is characterized with a series of developmental reprograming in root tissues, including extensive proliferation of cortical cells. We examined a possible involvement of the target of rapamycin (TOR) pathway, a central regulator of cell growth and proliferation in animals and yeasts, during soybean nodule development. Our results show that transcription of both GmTOR and its key downstream effector, GmS6K1, are activated during nodulation, which is paralleled with higher kinase activities of these gene products as well. RNAi-mediated knockdown of GmS6K1 impaired the nodule development with severely reduced nodule weight and numbers. In addition, expression of a few nodulins including leghemoglobin was also decreased, and consequently nitrogen fixation was found to be reduced by half. Proteomic analysis of the GmS6K1-RNAi nodules identified glutamine synthetase (GS), an essential enzyme for nitrogen assimilation in nodules, as one of the proteins that are significantly down regulated. These results appear to provide solid evidence for a functional link between GmS6K1 and nodule development.