Genomic Regions Research Articles

Recruitment of homodimeric proneural factors by conserved CAT-CAT E-boxes drives major epigenetic reconfiguration in cortical neurogenesis.

Proneural factors of the basic helix-loop-helix family coordinate neurogenesis and neurodifferentiation. Among them, NEUROG2 and NEUROD2 subsequently act to specify neurons of the glutamatergic lineage. Disruption of these factors, their target genes and binding DNA motifs has been linked to various neuropsychiatric disorders. Proneural factors bind to specific DNA motifs called E-boxes (hexanucleotides of the form CANNTG, composed of two CAN half sites on opposed strands). While corticogenesis heavily relies on E-box activity, the collaboration of proneural factors on different E-box types and their chromatin remodeling mechanisms remain largely unknown. Here, we conducted a comprehensive analysis using chromatin immunoprecipitation followed bysequencing (ChIP-seq) data for NEUROG2 and NEUROD2, along with time-matched single-cell RNA-seq, ATAC-seq and DNA methylation data from the developing mouse cortex. Our findings show that these factors are highly enriched in transiently active genomic regions during intermediate stages of neuronal differentiation. Although they primarily bind CAG-containing E-boxes, their binding in dynamic regions is notably enriched in CAT-CAT E-boxes (i.e. CATATG, denoted as 5'3' half sites for dimers), which undergo significant DNA demethylation and exhibit the highest levels of evolutionary constraint. Aided by HT-SELEX data reanalysis, structural modeling and DNA footprinting, we propose that these proneural factors exert maximal chromatin remodeling influence during intermediate stages of neurogenesis by binding as homodimers to CAT-CAT motifs. This study provides an in-depth integrative analysis of the dynamic regulation of E-boxes during neuronal development, enhancing our understanding of the mechanisms underlying the binding specificity of critical proneural factors.

CHARM and EvoETR: Precision epigenetic tools for gene silencing.

With the advent of gene editing technologies like CRISPR/Cas9, it has become possible to edit genomic regions of interest for research and therapeutic purposes. These technologies have also been adapted to alter gene expression without changing their DNA sequence, allowing epigenetic edits. While genetic editors make edits by cutting the genome at specified regions, epigenetic editors leverage the same targeting mechanism but act based on the epigenetic modifier fused to them, such as a methyltransferase. Here, we discuss two recently employed epigenetic editors (epi-editors) that silenced target genes involved in disease to mitigate their effects. Neumann etal. reported the construction of an epigenetic editor called CHARM that could methylate and silence the prion gene in mouse brains and subsequently switch itself off. Additionally, Capelluti etal. developed an epi-editor called EvoETR that knocked down Pcsk9 in the murine liver to reduce LDL levels. We aim to highlight the design principles underlying the design of these epi-editors to inform future editor designs.

Human Impacts on Great Lakes Walleye Sander vitreus Structure, Diversity and Local Adaptation.

Artificial propagation and wild release may influence the genetic integrity of wild populations. This practice has been prevalent in fisheries for centuries and is often termed 'stocking'. In the Laurentian Great Lakes (Great Lakes here-on), walleye populations faced declines from the 1950s to the 1970s, prompting extensive stocking efforts for restoration. By the mid-2010s, walleye populations showed signs of recovery, but the genetic legacy of stocking on population structure at the genomic level remains unclear. Using a dataset of 45,600 genome-aligned SNP loci genotyped in 1075 walleye individuals, we investigated the genetic impacts of over 50 years of stocking across the Great Lakes. Population structure was associated with both natural geographic barriers and stocking from non-native sources. Admixture between Lake Erie walleye and walleye from the re-populated Tittabawassee River indicate that stocking may have re-distributed putatively adaptive alleles around the Great Lakes. Genome scans identified FST outliers and evidence of selective sweeps, indicating local adaptation of spawning populations is likely. Notably, one genomic region showed strong differentiation between Muskegon River and walleye from the Tittabawassee River, which was re-populated by Muskegon strain walleye, suggesting admixture and selection both impact the observed genetic diversity. Overall, our study underscores how artificial propagation and translocations can significantly alter the evolutionary trajectory of populations. The findings highlight the complex interplay between stocking practices and population genetic diversity, emphasising the need for careful management strategies to preserve the genetic integrity of wild populations amidst conservation efforts.

Redefining copy number variation and single-nucleotide polymorphism counting via novel concepts based on recent PCR enhancements.

Human genes have numerous copy number variations (CNVs) and single-nucleotide polymorphisms (SNPs) that control most of the body’s core functions. On average, 12–16% of human genes have CNVs, and a single gene can have a few hundred to several thousand SNPs. Numerous genome-wide association studies (GWAS) have shown that CNVs and SNPs can coexist in certain genomic regions, amplifying their effects on gene expression and regulation and disease susceptibility. Researchers initially categorized CNVs and SNPs into two types: homozygous and heterozygous. However, copy numbers were soon found to have a much wider range, underscoring their significance in certain diseases and microbial interactions. Because of the significant impact of CNVs and SNPs, research groups worldwide have eagerly sought effective methods for detecting both simultaneously. Despite yielding some minor results, these simultaneous counting methods have failed to meet expectations, leaving researchers to measure CNVs and SNPs separately. To overcome these limitations, we developed a novel approach by combining primers designed using the STexS method with matching probes used in the STexS II method. This method successfully detected both CNVs and SNPs in CYP2A6 and CYP2A7 using a single quantitative polymerase chain reaction. Once properly adjusted based on the three core principles, this new method markedly improved the time, cost-effectiveness, and overall accuracy of determining an individual’s genetic status. Further testing of 100 human genomic DNA samples enabled calculations of the overall frequency of the [T] and [G] alleles of the CYP2A6 -48T>G SNP within an East Asian population yielded results that were highly congruent with those in a National Institutes of Health (NIH) database. This novel method will redefine genetic profiling and provide a means to successfully predict genetic characteristics and enhance personalized medicine by pinpointing appropriate individualized treatments.

Identification of complex Plasmodium falciparum genetic backgrounds circulating in Africa: a multicountry genomic epidemiology analysis

The population structure of the malaria parasite Plasmodium falciparum can reveal underlying adaptive evolutionary processes. Selective pressures to maintain complex genetic backgrounds can encourage inbreeding, producing distinct parasite clusters identifiable by population structure analyses. We analysed population structure in 3783 Pfalciparum genomes from 21countries across Africa, provided by the MalariaGEN Pf7 dataset. We used Principal Coordinate Analysis to cluster parasites, identity by descent (IBD) methods to identify genomic regions shared by cluster members, and linkage analyses to establish their co-inheritance patterns. Structural variants were reconstructed by de novo assembly and verified by long-read sequencing. We identified a strongly differentiated cluster of parasites, named AF1, comprising 47 (1·2%) of 3783samples analysed, distributed over 13countries across Africa, at locations over 7000km apart. Members of this cluster share a complex genetic background, consisting of up to 23loci harbouring many highly differentiated variants, rarely observed outside the cluster. IBD analyses revealed common ancestry at these loci, irrespective of sampling location. Outside the shared loci, however, AF1 members appear to outbreed with sympatric parasites. The AF1 differentiated variants comprise structural variations, including a gene conversion involving the dblmsp and dblmsp2 genes, and numerous single nucleotide polymorphisms. Several of the genes harbouring these mutations are functionally related, often involved in interactions with red blood cells including invasion, egress, and erythrocyte antigen export. We propose that AF1 parasites have adapted to some unidentified evolutionary niche, probably involving interactions with host erythrocytes. This adaptation involves a complex compendium of interacting variants that are rarely observed in Africa, which remains mostly intact despite recombination events. The term cryptotype was used to describe a common background interspersed with genomic regions of local origin. Bill & Melinda Gates Foundation.

Resolution of ring chromosomes, Robertsonian translocations, and complex structural variants from long-read sequencing and telomere-to-telomere assembly.

Delineation of structural variants (SVs) at sequence resolution in highly repetitive genomic regions has long been intractable. The sequence properties, origins, and functional effects of classes of genomic rearrangements such as ring chromosomes and Robertsonian translocations thus remain unknown. To resolve these complex structures, we leveraged several recent milestones in the field, including (1) the emergence of long-read sequencing, (2) the gapless telomere-to-telomere (T2T) assembly, and (3) a tool (BigClipper) to discover chromosomal rearrangements from long reads. We applied these technologies across 13 cases with ring chromosomes, Robertsonian translocations, and complex SVs that were unresolved by short reads, followed by validation using optical genome mapping (OGM). Our analyses resolved 10 of 13 cases, including a Robertsonian translocation and all ring chromosomes. Multiple breakpoints were localized to genomic regions previously recalcitrant to sequencing such as acrocentric p-arms, ribosomal DNA arrays, and telomeric repeats, and involved complex structures such as a deletion-inversion and interchromosomal dispersed duplications. We further performed methylation profiling from long-read data to discover phased differential methylation in a gene promoter proximal to a ring fusion, suggesting a long-range position effect (LRPE) with heterochromatin spreading. Breakpoint sequences suggested mechanisms of SV formation such as microhomology-mediated and non-homologous end-joining, as well as non-allelic homologous recombination. These methods provide some of the first glimpses into the sequence resolution of Robertsonian translocations and illuminate the structural diversity of ring chromosomes and complex chromosomal rearrangements with implications for genome biology, prediction of LRPEs from integrated multi-omics technologies, and molecular diagnostics in rare disease cases.

Patterns of the within-host evolution of human norovirus in immunocompromised individuals and implications for treatment

Currently, there is no licensed treatment for chronic norovirus infections, but the use of intra-duodenally-delivered immunoglobulins is promising; nevertheless, varying results have limited their wide use. Little is known about the relationship between norovirus genetic diversity and treatment efficacy. We analyzed the norovirus within-host diversity and evolution in a cohort of 20 immunocompromised individuals using next-generation sequencing (NGS) and clone-based sequencing of the capsid (VP1) gene. Representative VP1s were expressed and their glycan receptor binding affinity and antigenicity were evaluated. The P2 domain, within the VP1, accumulated up to 30-fold more non-synonymous mutations than other genomic regions. Intra-host virus populations in these patients tended to evolve into divergent lineages that were often antigenically distinct. Several of these viruses were widely resistant to binding-blocking antibodies in immunoglobulin preparations. Notably, for one patient, a single amino-acid substitution in the P2 domain resulted in an immune-escape phenotype, and it was likely the main contributor to treatment failure. Furthermore, we found evidence for transmission of late-stage viruses between two immunocompromised individuals. The findings demonstrated that within-host noroviruses in chronic infections tend to evolve into antigenically distinct subpopulations. This antigenic evolution was likely caused by the remaining low immunity levels exerted by immunocompromised individuals, possibly undermining antiviral treatment. Our observations provide insights into norovirus (within-host) evolution and treatment. Erasmus MC grant mRACE, the European Union's Horizon 2020 research and innovation program under grant agreement No. 874735 (VEO), and the NWO STEVIN award (Koopmans).

Identification of candidate genes and genomic prediction for early heifer pregnancy in Nelore beef cattle

This study aimed to assess the influence of differential weighting in genomic regions harboring candidate causal loci on the prediction accuracy and inflation for early heifer pregnancy (heifers that calved up to 30 months of age) in Nellore (Bos indicus) heifers using the single step genomic BLUP model (ssGBLUP). Phenotypic records of 102,294 Nellore heifers born between 2010 and 2017 were used in this study. The pedigree dataset harbored information from 176,107 animals born between 1998 and 2017, including 5,145 sires and 35,705 dams. Seven different models for genomic prediction were defined by combining the SNP weights obtained in the iterations (1st and 2nd) of the weighted single step GWAS (ssw1GBLUP and ssw2GBLUP) or candidate QTLs reported in the literature. Hence, the lambda (λ) values estimated in the WssGWAS were used to weight the SNPs adjacent to the candidate regions or QTL previously reported in the literature. To estimate the genetic parameters and perform the WssGWAS and WssGBLUP for early heifer pregnancy, a single-trait Bayesian analysis considering a threshold animal model was used. Accuracy, bias, and inflation parameters were evaluated in the validation subset based on the linear regression (LR) method. Genomic windows of ten consecutive SNPs that explained more than 0.5% of the additive genetic variance were selected to explore and determine possible candidate genes. Among the identified genes, we can highlight the PGRMC2, TENM3, GRIP1, TMEM45A, and KLF3, given their roles in endocrine fertility, expression of contractile proteins, average daily gain, dry matter intake, and fat deposition. Several genomic regions associated with QTL related to early heifer pregnancy were identified. The identification of such regions and the respective candidate genes associated with sexual precocity and fertility would contribute to improve the genetic knowledge regarding early sexual precocity of Nellore cattle. The prediction accuracy increased roughly 25.6% using the ssGBLUP compared to BLUP models. The prediction accuracy with the WssGBLUP when incorporating weighted SNPs with the λ values obtained in the 1st (ssw1GBLUP) and 2nd iteration (ssw2GBLUP) of the WssGWAS was higher (∼18%) than that described for the ssGBLUP model. The inflation also increased the weighting of the most relevant SNPs obtained with the GWAS, most likely overestimating the GEBV. The models that weighted SNPs close to QTLs reported in the literature yielded to less biased and deflated predictions compared to ssw1GBLUP and ssw2GBLUP models. Genomic selection is a feasible alternative for genomic evaluation of early heifer pregnancy in Nellore beef cattle by increasing the prediction accuracy of young animals. In addition, the use of information obtained from the WssGWAS is an alternative to increase reliability and reduce genomic prediction bias. Therefore, the results obtained herein indicate that it is possible to improve the prediction accuracy and reduce the bias of genomic prediction by using genomic information and differentially weighted genomic regions harboring candidate QTLs previously reported in the literature for early heifer pregnancy.

ROR1-AS1: A Meaningful Long Noncoding RNA in Oncogenesis.

Long noncoding RNA (lncRNA) is a non-coding RNA with a length of more than 200 nucleotides, involved in multiple regulatory processes in vivo, and is related to the physiology and pathology of human diseases. An increasing number of experimental results suggest that when lncRNA is abnormally expressed, it results in the development of tumors. LncRNAs can be divided into five broad categories: sense, antisense, bidirectional, intronic, and intergenic. Studies have found that some antisense lncRNAs are involved in a variety of human tumorigenesis. The newly identified ROR1-AS1, which functions as an antisense RNA of ROR1, is located in the 1p31.3 region of the human genome. Recent studies have reported that abnormal expression of lncRNA ROR1-AS1 can affect cell growth, proliferation, invasion, and metastasis and increase oncogenesis and tumor spread, indicating lncRNA ROR1-AS1 as a promising target for many tumor biological therapies. In this study, the pathophysiology and molecular mechanism of ROR1-AS1 in various malignancies are discussed by retrieving the related literature. ROR1-AS1 is a cancer-associated lncRNA, and studies have found that it is either over- or underexpressed in multiple malignancies, including liver cancer, colon cancer, osteosarcoma, glioma, cervical cancer, bladder cancer, lung adenocarcinoma, and mantle cell lymphoma. Furthermore, it has been demonstrated that lncRNA ROR1-AS1 participates in proliferation, migration, invasion, and suppression of apoptosis of cancer cells. Furthermore, lncRNA ROR1-AS1 promotes the development of tumors by up-regulating or downregulating ROR1-AS1 conjugates and various pathways and miR-504, miR-4686, miR-670-3p, and miR-375 sponges, etc., suggesting that lncRNA ROR1-AS1 may be used as a marker in tumors or a potential therapeutic target for a variety of tumors.

Development and evolution of Drosophila chromatin landscape in a 3D genome context

Little is known about how the epigenomic states change during development and evolution in a 3D genome context. Here we use Drosophila pseudoobscura with complex turnover of sex chromosomes as a model to address this, by collecting massive epigenomic and Hi-C data from five developmental stages and three adult tissues. We reveal that over 60% of the genes and transposable elements (TE) exhibit at least one developmental transition of chromatin state. Transitions on specific but not housekeeping enhancers are associated with specific chromatin loops and topologically associated domain borders (TABs). While evolutionarily young TEs are generally silenced, old TEs more often have been domesticated as interacting TABs or specific enhancers. But on the recently evolved X chromosome, young TEs are instead often active and recruited as TABs, due to acquisition of dosage compensation. Overall we characterize how Drosophila epigenomic landscapes change during development and in response to chromosome evolution, and highlight the important roles of TEs in genome organization and regulation.

Network and systems biology approaches help investigate gene regulatory interactions between Salmonella disease and host in chickens: Model-based in silico evidence combined with gene expression assays.

Salmonella enteritidis (SE), a previously widespread infectious disease, is still cited as a major factor in economic losses in commercial chicken production. The host's genetic immune system determines the pathogenicity of a particular bacterium. To shed light on this topic, it was necessary to understand the key candidate genes essential for regulating susceptibility and resistance to the target disease. The field of poultry farming in particular has benefited greatly from the connection between quantitative and molecular genetics. This study aims to identify the most important immune-related genes and their signalling pathways (gene ontology, co-expression and interactions) and to analyse their accumulation in host-resistant SE diseases by combining gene expression assays with model-based in silico evidence. A two-step experimental design is followed. To start, we used free computational tools and online bioinformatics resources, including predicting gene function using a multiple association network integration algorithm (geneMania), the Kyoto Encyclopedia of Genes and Genomes, the Annotation, Visualization and Integrated Discovery (DAVID) database and the stimulator of interferon genes. Natural resistance-associated macrophage protein 1 (NRAMP1), Toll-like receptor 4 (TLR4), interferon-γ (IFNγ), immunoglobulin Y (IgY) and interleukin 8 (IL8) were among the five genes whose expression levels in liver, spleen, and cecum were evaluated at 1107 SE after 48h of inoculation. This molecular study was developed in the second phase of research to validate the in silico observations. Next, we use five promising biomarkers for relative real-time polymerase chain reaction (PCR) quantification: TLR4, IL8, NRAMP1, IFNγ and IgY genes in two case and control assays. The 2-∆∆Ct Livak and Schmittgen method was used to compare the expression of genes in treated and untreated samples. This method normalizes the expression of the target gene to that of actin, an internal control and estimates the change in expression relative to the untreated control. Internal control was provided by the Beta actin gene. Next, statistically, the postdoc test was used for the evaluation of treatments using SAS version 9.4, and p values of 0.05 and 0.01 were chosen for significant level. Interestingly, the results of our study suggest the involvement of various factors in the host immune response to Salmonella. These include inducible nitric oxide synthase, NRAMP1, immunoglobulin light chain (IgL), transforming growth factor B family (TGFb2, TGFb3 and TGFb4), interleukin 2 (IL2), apoptosis inhibitor protein 1 (IAP1), TLR4, myeloid differentiation protein 2 (MD2), IFNγ, caspase 1 (CASP1), lipopolysaccharide-induced tumour necrosis factor (LITAF), cluster of differentiation 28 (CD28) and prosaposin (PSAP). The summary of gene ontology and related genes found for SE resistance was surprisingly comprehensive and covered the following topics: positive regulation of endopeptidase activity, interleukin-8 production, chemokine production, interferon-gamma production, interleukin-6 production, positive regulation of mononuclear cell proliferation and response to interferon-gamma. The role of these promising biomarkers in our networks against SE susceptibility is essentially confirmed by these results. After 48h, the spleen showed significant expression of the tissue-specific gene expression patterns for NRAMP1 and IL8 in the cecum, spleen and liver. Based on this information, this report searches for resistance and susceptibility lineages in most genomic regions for SE. In conclusion, the development of an appropriate selection program to improve resistance to salmonellosis can be facilitated by a comprehensive understanding of the immune responses of the chicken immune system after SE exposure.

Historic breeding practices contribute to germplasm divergence in leaf specialized metabolism and ecophysiology in cultivated sunflower (Helianthus annuus).

The use of hybrid breeding systems to increase crop yields has been the cornerstone of modern agriculture and is exemplified in the breeding and improvement of cultivated sunflower (Helianthus annuus). However, it is poorly understood what effect supporting separate breeding pools in such systems, combined with continued selection for yield, may have on leaf ecophysiology and specialized metabolite variation. We analyzed 288 lines of cultivated H. annuus to examine the genomic basis of several specialized metabolites and agronomically important traits across major heterotic groups. Heterotic group identity supports phenotypic divergences between fertility restoring and cytoplasmic male-sterility maintainer lines in leaf ecophysiology and specialized metabolism. However, the divergence is not associated with physical linkage to nuclear genes that support current hybrid breeding practices in cultivated H. annuus. Additionally, we identified four genomic regions associated with leaf ecophysiology and specialized metabolism that colocalize with previously identified QTLs for quantitative self-compatibility traits and with S-protein homolog (SPH) proteins, a recently discovered family of proteins associated with self-incompatibility and self/nonself recognition in Papaver rhoeas (common poppy) with suggested conserved downstream mechanisms among eudicots. Further work is necessary to confirm the self-incompatibility mechanisms in cultivated H. annuus and their relationship to the integrative and polygenic architecture of leaf ecophysiology and specialized metabolism in cultivated sunflower. However, because self-compatibility is a derived quantitative trait in cultivated H. annuus, trait linkage to divergent phenotypic traits may have partially arisen as a potential unintended consequence of historical breeding practices and selection for yield.

Comprehensive evaluation of the impact of whole-genome bisulfite sequencing (WGBS) on the fragmentomic characteristics of plasma cell-free DNA

BackgroundCell-free DNA (cfDNA) is non-randomly fragmented in human body fluids. Analyzing such fragmentation patterns of cfDNA holds great promise for liquid biopsy. Whole-genome bisulfite sequencing (WGBS) is widely used for cfDNA methylation profiling. However, its applicability for studying fragmentomic characteristics remains largely unexplored. MethodsWe performed paired WGBS and whole-genome sequencing (WGS) on 66 peripheral plasma samples from 58 pregnant women. Then, we systematically compared the fragmentation patterns of cell-free nuclear DNA and mitochondrial DNA (mtDNA) sequenced from these two approaches. Additionally, we evaluated the extent of the size shortening in fetal-derived cfDNA and estimated the fetal DNA fraction in maternal plasma using both sequencing methods. ResultsCompared to WGS samples, WGBS samples demonstrated a significantly lower genome coverage and higher GC content in cfDNA. They also showed a significant decrease in the size of cell-free nuclear DNA, along with alterations in the end motif pattern that were specifically associated with CpG and “CC” sites. While there was a slight shift in the inferred nucleosome footprint from cfDNA coverages in WGBS samples, the cfDNA coverage patterns in CTCF and TSS regions remained highly consistent between these two sequencing methods. Both methods accurately reflected gene expression levels through their TSS coverages. Additionally, WGBS samples exhibited an increased abundance and longer length of mtDNA in plasma. Furthermore, we observed the size shortening of fetal cfDNA in plasma consistently, with a highly correlated fetal DNA fraction inferred by cfDNA coverage between WGBS and WGS samples (r = 0.996). However, the estimated fetal cfDNA fraction in WGBS samples was approximately 7 % lower than in WGS samples. ConclusionsWe confirmed that WGBS can introduce artificial breakages to cfDNA, leading to altered fragmentomic patterns in both nuclear and mitochondrial DNA. However, WGBS cfDNA remains suitable for analyzing certain cfDNA fragmentomic characteristics, such as coverage in genome regulation regions and the essential characteristics of fetal DNA in maternal plasma.

Genomic regions associated with embryonic loss in primiparous Holstein cows

IntroductionReproductive efficiency in dairy cattle bred by artificial insemination (AI) or via embryo transfer (ET) is important as a means of facilitating the rate of genetic improvement by utilizing superior genetics from sires and donor dams. Fertility is especially challenging in primiparous cows as they have recently calved, are still growing, and are lactating. Fertility, measured by the conception rate from the first service (CCR1), is the number of cows that become pregnant as a result of the first AI or ET compared to all cows serviced, whereas times bred (TBRD) is the number of inseminations cows bred by AI needed to achieve a successful pregnancy.MethodsThis study aimed to identify the loci associated with and gene sets enriched for the CCR1 in primiparous cows that were bred by AI (n = 882) or were ET recipients (n = 353), and to identify the loci associated with the TBRD (n= 1,851) in primiparous cows. A genome-wide association analysis and gene set enrichment analysis were performed using single-nucleotide polymorphism (SNP) data for the CCR1 and TBRD in primiparous cows. In cows bred by AI, two loci containing five positional candidate genes were associated with the CCR1 (p < 1 × 10−5) and one gene set with 18 leading-edge genes was enriched (NES ≥3) for the CCR1.Results and discussionIn cows that were ET recipients, five loci were associated with the CCR1, but no gene sets were enriched for the CCR1. For the second phenotype, five loci with nine positional candidate genes were associated with the TBRD and two gene sets with eight leading-edge genes were enriched for the TBRD. Identifying the loci associated with or gene sets enriched for the CCR1 and TBRD provides an opportunity to better understand embryonic loss in AI-bred and ET-recipient cows and to use this information to select for higher reproductive efficiency.

Deciphering the mystery of CHNG3.

Congenital hypothyroidism (CH), characterized by insufficient thyroid hormone production due to abnormalities in the hypothalamic-pituitary-thyroid axis, is the most common congenital endocrine disorder. We previously conducted comprehensive genetic screening of 102 patients with permanent CH born in Kanagawa Prefecture, Japan and identified mutations in several genes in 19 CH patients, including defects in genes encoding dual oxidase 2, thyroglobulin, thyrotropin receptor, thyroid peroxidase, and paired-box 8. Despite these findings, approximately 80% of cases remain unexplained. CH pedigrees unexplained by known genetic forms of CH have been reported in the literature and registered as congenital hypothyroidism, nongoitrous, 3 (CHNG3; %609893) in Online Mendelian Inheritance in Man. We also identified a Japanese pedigree of CH that was compatible with CHNG3. However, the exact genetic cause of CHNG3 was not revealed by standard analysis methods such as exome sequencing and array comparative genomic hybridization. We therefore took a combined approach and analyzed a total of 11 undiagnosed CH pedigrees by whole genome sequencing to analyze a 3-Mb linkage region, and found a disease-causing variant affecting a TTTG microsatellite in a noncoding region on chromosome 15. Further analysis revealed that 13.9% of 989 Japanese CH patients had abnormalities involving the TTTG microsatellite, with a substantial proportion (41.5%) of familial CH cases carrying these mutations. Identification of the genetic cause of CHNG3 provides new insights into the pathogenesis of CH, and highlights the need for continued exploration of noncoding genomic regions in Mendelian disorders of unknown etiology.

Discover QTLs for the level of blood components in Shaoxing duck using GWAS and haplotype sharing analysis

Blood composition is indicative of health-related traits such as immunity and metabolism. The use of molecular genetics to investigate alterations in these attributes in laying ducks is a novel approach. Our objective was to employ genome − wide association studies (GWAS) and haplotype − sharing analysis to identify genomic regions and potential genes associated with 11 blood components in Shaoxing ducks. Our findings revealed 35 SNPs and 1 SNP associated with low-density lipoprotein cholesterol (LDL) and globulin (GLB), respectively. We identified 36 putative candidate genes for the LDL trait in close proximity to major QTLs and key loci. Based on their biochemical and physiological properties, TRA2A, NPY, ARHGEF26, DHX36, and AADAC are the strongest putative candidate genes. Through linkage disequilibrium analysis and haplotype sharing analysis, we identified three haplotypes and one haplotype, respectively, that were significantly linked with LDL and GLB. These haplotypes could be selected as potential candidate haplotypes for molecular breeding of Shaoxing ducks. Additionally, we utilized a bootstrap test to verify the reliability of GWAS with small experimental samples. The test can be accessed at https://github.com/xuwenwu24/Bootstrap-test.

FindGSEP: estimating genome size of polyploid species using k-mer frequencies.

Estimating genome size using k-mer frequencies, which plays a fundamental role in designing genome sequencing and analysis projects, has remained challenging for polyploid species, i.e., ploidy p > 2. To address this, we introduce "findGSEP," which is designed based on iterative curve fitting of k-mer frequencies. Precisely, it first disentangles up to p normal distributions by analyzing k-mer frequencies in whole genome sequencing of the focal species. Second, it computes the sizes of genomic regions related to 1∼p (homologous) chromosome(s) using each respective curve fitting, from which it infers the full polyploid and average haploid genome size. "findGSEP" can handle any level of ploidy p, and infer more accurate genome size than other well-known tools, as shown by tests using simulated and real genomic sequencing data of various species including octoploids. "findGSEP" was implemented as a web server, which is freely available at http://146.56.237.198:3838/findGSEP/. Also, "findGSEP" was implemented as an R package for parallel processing of multiple samples. Source code and tutorial on its installation and usage is available at https://github.com/sperfu/findGSEP.

Advancing CRISPR-Based Solutions for COVID-19 Diagnosis and Therapeutics.

Since the onset of the COVID-19 pandemic, a variety of diagnostic approaches, including RT-qPCR, RAPID, and LFA, have been adopted, with RT-qPCR emerging as the gold standard. However, a significant challenge in COVID-19 diagnostics is the wide range of symptoms presented by patients, necessitating early and accurate diagnosis for effective management. Although RT-qPCR is a precise molecular technique, it is not immune to false-negative results. In contrast, CRISPR-based detection methods for SARS-CoV-2 offer several advantages: they are cost-effective, time-efficient, highly sensitive, and specific, and they do not require sophisticated instruments. These methods also show promise for scalability, enabling diagnostic tests. CRISPR technology can be customized to target any genomic region of interest, making it a versatile tool with applications beyond diagnostics, including therapeutic development. The CRISPR/Cas systems provide precise gene targeting with immense potential for creating next-generation diagnostics and therapeutics. One of the key advantages of CRISPR/Cas-based therapeutics is the ability to perform multiplexing, where different sgRNAs or crRNAs can target multiple sites within the same gene, reducing the likelihood of viral escape mutants. Among the various CRISPR systems, CRISPR/Cas13 and CARVER (Cas13-assisted restriction of viral expression and readout) are particularly promising. These systems can target a broad range of single-stranded RNA viruses, making them suitable for the diagnosis and treatment of various viral diseases, including SARS-CoV-2. However, the efficacy and safety of CRISPR-based therapeutics must be thoroughly evaluated in pre-clinical and clinical settings. While CRISPR biotechnologies have not yet been fully harnessed to control the current COVID-19 pandemic, there is an optimism that the limitations of the CRISPR/Cas system can be overcome soon. This review discusses how CRISPR-based strategies can revolutionize disease diagnosis and therapeutic development, better preparing us for future viral threats.

Epigenetic Study of Cohort of Monozygotic Twins With Hypertrophic Cardiomyopathy Due to MYBPC3 (Cardiac Myosin-Binding Protein C).

Hypertrophic cardiomyopathy is an autosomal dominant cardiac disease. The mechanisms that determine its variable expressivity are poorly understood. Epigenetics could play a crucial role in bridging the gap between genotype and phenotype by orchestrating the interplay between the environment and the genome regulation. In this study we aimed to establish a possible correlation between the peripheral blood DNA methylation patterns and left ventricular hypertrophy severity in patients with hypertrophic cardiomyopathy, evaluating the potential impact of lifestyle variables and providing a biological context to the observed changes. Methylation data were obtained from peripheral blood samples (Infinium MethylationEPIC BeadChip arrays). We employed multiple pair-matched models to extract genomic positions whose methylation correlates with the degree of left ventricular hypertrophy in 3 monozygotic twin pairs carrying the same founder pathogenic variant (MYBPC3 p.Gly263Ter). This model enables the isolation of the environmental influence, beyond age, on DNA methylation changes by removing the genetic background. Our results revealed a more anxious personality among more severely affected individuals. We identified 56 differentially methylated positions that exhibited moderate, proportional changes in methylation associated with left ventricular hypertrophy. These differentially methylated positions were enriched in regions regulated by repressor histone marks and tended to cluster at genes involved in left ventricular hypertrophy development, such as HOXA5, TRPC3, UCN3, or PLSCR2, suggesting that changes in peripheral blood may reflect myocardial alterations. We present a unique pair-matched model, based on 3 monozygotic twin pairs carrying the same founder pathogenic variant and different phenotypes. This study provides further evidence of the pivotal role of epigenetics in hypertrophic cardiomyopathy variable expressivity.

Number of human protein interactions correlates with structural, but not regulatory conservation of the respective genes.

The differential ratio of nonsynonymous to synonymous nucleotide substitutions (dN/dS) is a common measure of the rate of structural evolution in proteincoding genes. In addition, we recently suggested that the proportion of transposable elements in gene promoters that host functional genomic sites serves as a marker of the rate of regulatory evolution of genes. Such functional genomic regions may include transcription factor binding sites and modified histone binding loci. Here, we constructed a model of the human interactome based on 600,136 documented molecular interactions and investigated the overall relationship between the number of interactions of each protein and the rate of structural and regulatory evolution of the corresponding genes. By evaluating a total of 4,505 human genes and 1,936 molecular pathways we found a general correlation between structural and regulatory evolution rate metrics (Spearman 0.08-0.16 and 0.25-0.37 for gene and pathway levels, respectively, p < 0.01). Further exploration revealed in the established human interactome model lack of correlation between the rate of gene regulatory evolution and the number of protein interactions on gene level, and weak negative correlation (∼0.15) on pathway level. We also found a statistically significant negative correlation between the rate of gene structural evolution and the number of protein interactions (Spearman -0.11 and -0.3 for gene and pathway levels, respectively, p < 0.01). Our result suggests stronger structural rather than regulatory conservation of genes whose protein products have multiple interaction partners.