Single Allele Research Articles

The John Charnley Award: The Impact of Human Leukocyte Antigen Genotype on Bacterial Infection Rates and Successful Eradication in Total Hip Arthroplasty

BackgroundGenetics play an important role in several medical domains; however, the influence of human leukocyte antigen (HLA) genotype on the development of periprosthetic joint infection (PJI) in total hip arthroplasty (THA) remains unknown. The primary aim of this study was to determine if HLA genotype is associated with the development of bacterial PJI in THA. Secondarily, we evaluated the association between HLA genotype and PJI treatment success. MethodsA retrospective, matched, case-control study was performed using prospectively collected data from a single institution. A total of 49 patients who underwent primary THA were included, with a mean follow-up of 8.5 years (range, 4.2 to 12.9). The 23 cases (PJI) and 26 controls (no PJI) were matched for age, sex, follow-up, body mass index, primary diagnosis, and comorbidities (P > .05). High-resolution genetic analysis targeting 11 separate HLA loci was performed in all patients using serum samples. The HLA gene frequencies and carriage rates were determined and compared between cohorts. A subgroup analysis of PJI treatment success (18) and failure (5) was performed. Statistical significance was set at P = .10 for genetic analysis and at 0.05 for all other analyses. ResultsThere were 4 HLA alleles that were significantly associated with the development of PJI. The 3 at-risk alleles included HLA-C∗06:02 (odds ratio 5.25, 95% CI [confidence interval] 0.96 to 28.6, P = .064), HLA-DQA1∗04:01 (P = .096), and HLA-DQB1∗04:02 (P = .096). The single protective allele was HLA-C∗03:04 (odds ratio 0.12, 95% CI 0.01 to 1.10, P = .052). There were no specific HLA alleles that were associated with treatment success or failure. ConclusionsThis study suggests that there are at-risk and protective HLA alleles associated with the development of PJI in THA. To our knowledge, this is the first study to demonstrate an association between patient HLA genotype and the development of PJI. A larger study of the subject matter is necessary and warranted.

Semaphorin-3A regulates liver sinusoidal endothelial cell porosity and promotes hepatic steatosis

Prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as non-alcoholic fatty liver disease, increases worldwide and associates with type 2 diabetes and other cardiometabolic diseases. Here we demonstrate that Sema3a is elevated in liver sinusoidal endothelial cells of animal models for obesity, type 2 diabetes and MASLD. In primary human liver sinusoidal endothelial cells, saturated fatty acids induce expression of SEMA3A, and loss of a single allele is sufficient to reduce hepatic fat content in diet-induced obese mice. We show that semaphorin-3A regulates the number of fenestrae through a signaling cascade that involves neuropilin-1 and phosphorylation of cofilin-1 by LIM domain kinase 1. Finally, inducible vascular deletion of Sema3a in adult diet-induced obese mice reduces hepatic fat content and elevates very low-density lipoprotein secretion. Thus, we identified a molecular pathway linking hyperlipidemia to microvascular defenestration and early development of MASLD.

Establishment and maintenance of random monoallelic expression.

This Review elucidates the regulatory principles of random monoallelic expression by focusing on two well-studied examples: the X-chromosome inactivation regulator Xist and the olfactory receptor gene family. Although the choice of a single X chromosome or olfactory receptor occurs in different developmental contexts, common gene regulatory principles guide monoallelic expression in both systems. In both cases, an event breaks the symmetry between genetically and epigenetically identical copies of the gene, leading to the expression of one single random allele, stabilized through negative feedback control. Although many regulatory steps that govern the establishment and maintenance of monoallelic expression have been identified, key pieces of the puzzle are still missing. We provide an overview of the current knowledge and models for the monoallelic expression of Xist and olfactory receptors. We discuss their similarities and differences, and highlight open questions and approaches that could guide the study of other monoallelically expressed genes.

Sub-microscopic Plasmodium falciparum infections and multiple drug resistant single nucleotide polymorphic alleles in pregnant women from southwestern Nigeria

ObjectivesThe study evaluated sub-microscopic malaria infections in pregnancy using two malaria Rapid Diagnostic Tests (mRDTs), microscopy and RT-PCR and characterized Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and Plasmodium falciparum dihydropteroate synthase (Pfdhps) drug resistant markers in positive samples.MethodsThis was a cross sectional survey of 121 pregnant women. Participants were finger pricked, blood drops were collected for rapid diagnosis with P. falciparum histidine-rich protein 11 rapid diagnostic test kit and the ultra-sensitive Alere Pf malaria RDT, Blood smears for microscopy and dried blood spots on Whatman filter paper for molecular analysis were made. Real time PCR targeting the var acidic terminal sequence (varATS) gene of P. falciparum was carried out on a CFX 96 real time system thermocycler (BioRad) in discriminating malaria infections. For each run, laboratory strain of P. falciparum 3D7 and nuclease free water were used as positive and negative controls respectively. Additionally, High resolution melt analyses was employed for genotyping of the different drug resistance markers.ResultsOut of one hundred and twenty-one pregnant women sampled, the SD Bioline™ Malaria Ag P.f HRP2-based malaria rapid diagnostic test (mRDT) detected eight (0.06%) cases, the ultra-sensitive Alere™ malaria Ag P.f rapid diagnostic test mRDT had similar outcome in the same samples as detected by the HRP2-based mRDT. Microscopy and RT-PCR confirmed four out of the eight infections detected by both rapid diagnostic tests as true positive and RT-PCR further detected three false negative samples by the two mRDTs providing a sub-microscopic malaria prevalence of 3.3%. Single nucleotide polymorphism in Pfdhps gene associated with sulphadoxine resistance revealed the presence of S613 mutant genotypes in three of the seven positive isolates and isolates with mixed wild/mutant genotype at codon A613S. Furthermore, four mixed genotypes at the A581G codon were also recorded while the other Pfdhps codons (A436G, A437G and K540E) showed the presence of wild type alleles. In the Pfdhfr gene, there were mutations in 28.6%, 28.6%, and 85.7% at the I51, R59 and N108 codons respectively. Mixed wild and mutant type genotypes were also observed in 28.6% each of the N51I, and C59R codons. For the Pfcrt, two haplotypes CVMNK and CVIET were observed. The SVMNT was altogether absent. Triple mutant CVIET 1(14.3%) and triple mutant + wild genotype CVIET + CVMNK 1(14.3%) were observed. The Pfmdr1 haplotypes were single mutants YYND 1(14.3%); NFND 1(14.3%) and double mutants YFND 4(57.1%); YYDD 1(14.3%).

FASTMAP-a flexible and scalable immunopeptidomics pipeline for HLA- and antigen-specific T-cell epitope mapping based on artificial antigen-presenting cells.

The study of peptide repertoires presented by major histocompatibility complex (MHC) molecules and the identification of potential T-cell epitopes contribute to a multitude of immunopeptidome-based treatment approaches. Epitope mapping is essential for the development of promising epitope-based approaches in vaccination as well as for innovative therapeutics for autoimmune diseases, infectious diseases, and cancer. It also plays a critical role in the immunogenicity assessment of protein therapeutics with regard to safety and efficacy concerns. The main challenge emerges from the highly polymorphic nature of the human leukocyte antigen (HLA) molecules leading to the requirement of a peptide mapping strategy for a single HLA allele. As many autoimmune diseases are linked to at least one specific antigen, we established FASTMAP, an innovative strategy to transiently co-transfect a single HLA allele combined with a disease-specific antigen into a human cell line. This approach allows the specific identification of HLA-bound peptides using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Using FASTMAP, we found a comparable spectrum of endogenous peptides presented by the most frequently expressed HLA alleles in the world's population compared to what has been described in literature. To ensure a reliable peptide mapping workflow, we combined the HLA alleles with well-known human model antigens like coagulation factor VIII, acetylcholine receptor subunit alpha, protein structures of the SARS-CoV-2 virus, and myelin basic protein. Using these model antigens, we have been able to identify a broad range of peptides that are in line with already published and in silico predicted T-cell epitopes of the specific HLA/model antigen combination. The transient co-expression of a single affinity-tagged MHC molecule combined with a disease-specific antigen in a human cell line in our FASTMAP pipeline provides the opportunity to identify potential T-cell epitopes/endogenously processed MHC-bound peptides in a very cost-effective, fast, and customizable system with high-throughput potential.

Zygosity-based sex determination in a butterfly drives hypervariability of Masculinizer.

Nature has devised many ways of producing males and females. Here, we report on a previously undescribed mechanism for Lepidoptera that functions without a female-specific gene. The number of alleles or allele heterozygosity in a single Z-linked gene (BaMasc) is the primary sex-determining switch in Bicyclus anynana butterflies. Embryos carrying a single BaMasc allele develop into WZ (or Z0) females, those carrying two distinct alleles develop into ZZ males, while (ZZ) homozygotes initiate female development, have mismatched dosage compensation, and die as embryos. Consequently, selection against homozygotes has favored the evolution of spectacular allelic diversity: 205 different coding sequences of BaMasc were detected in a sample of 246 females. The structural similarity of a hypervariable region (HVR) in BaMasc to the HVR in Apis mellifera csd suggests molecular convergence between deeply diverged insect lineages. Our discovery of this primary switch highlights the fascinating diversity of sex-determining mechanisms and underlying evolutionary drivers.

Differences in 5'untranslated regions highlight the importance of translational regulation of dosage sensitive genes

BackgroundUntranslated regions (UTRs) are important mediators of post-transcriptional regulation. The length of UTRs and the composition of regulatory elements within them are known to vary substantially across genes, but little is known about the reasons for this variation in humans. Here, we set out to determine whether this variation, specifically in 5’UTRs, correlates with gene dosage sensitivity.ResultsWe investigate 5’UTR length, the number of alternative transcription start sites, the potential for alternative splicing, the number and type of upstream open reading frames (uORFs) and the propensity of 5’UTRs to form secondary structures. We explore how these elements vary by gene tolerance to loss-of-function (LoF; using the LOEUF metric), and in genes where changes in dosage are known to cause disease. We show that LOEUF correlates with 5’UTR length and complexity. Genes that are most intolerant to LoF have longer 5’UTRs, greater TSS diversity, and more upstream regulatory elements than their LoF tolerant counterparts. We show that these differences are evident in disease gene-sets, but not in recessive developmental disorder genes where LoF of a single allele is tolerated.ConclusionsOur results confirm the importance of post-transcriptional regulation through 5'UTRs in tight regulation of mRNA and protein levels, particularly for genes where changes in dosage are deleterious and lead to disease. Finally, to support gene-based investigation we release a web-based browser tool, VuTR, that supports exploration of the composition of individual 5'UTRs and the impact of genetic variation within them.

Reversal of C9orf72 mutation-induced transcriptional dysregulation and pathology in cultured human neurons by allele-specific excision

Efforts to genetically reverse C9orf72 pathology have been hampered by our incomplete understanding of the regulation of this complex locus. We generated five different genomic excisions at the C9orf72 locus in a patient-derived induced pluripotent stem cell (iPSC) line and a non-diseased wild-type (WT) line (11 total isogenic lines), and examined gene expression and pathological hallmarks of C9 frontotemporal dementia/amyotrophic lateral sclerosis in motor neurons differentiated from these lines. Comparing the excisions in these isogenic series removed the confounding effects of different genomic backgrounds and allowed us to probe the effects of specific genomic changes. A coding single nucleotide polymorphism in the patient cell line allowed us to distinguish transcripts from the normal vs. mutant allele. Using digital droplet PCR (ddPCR), we determined that transcription from the mutant allele is upregulated at least 10-fold, and that sense transcription is independently regulated from each allele. Surprisingly, excision of the WT allele increased pathologic dipeptide repeat poly-GP expression from the mutant allele. Importantly, a single allele was sufficient to supply a normal amount of protein, suggesting that the C9orf72 gene is haplo-sufficient in induced motor neurons. Excision of the mutant repeat expansion reverted all pathology (RNA abnormalities, dipeptide repeat production, and TDP-43 pathology) and improved electrophysiological function, whereas silencing sense expression did not eliminate all dipeptide repeat proteins, presumably because of the antisense expression. These data increase our understanding of C9orf72 gene regulation and inform gene therapy approaches, including antisense oligonucleotides (ASOs) and CRISPR gene editing.

Redundant and Distinct Roles of Two 14-3-3 Proteins in Fusarium sacchari, Pathogen of Sugarcane Pokkah Boeng Disease.

Fusarium sacchari, a key pathogen of sugarcane, is responsible for the Pokkah boeng disease (PBD) in China. The 14-3-3 proteins have been implicated in critical developmental processes, including dimorphic transition, signal transduction, and carbon metabolism in various phytopathogenic fungi. However, their roles are poorly understood in F. sacchari. This study focused on the characterization of two 14-3-3 protein-encoding genes, FsBmh1 and FsBmh2, within F. sacchari. Both genes were found to be expressed during the vegetative growth stage, yet FsBmh1 was repressed at the sporulation stage in vitro. To elucidate the functions of these genes, the deletion mutants ΔFsBmh1 and ΔFsBmh2 were generated. The ΔFsBmh2 exhibited more pronounced phenotypic defects, such as impaired hyphal branching, septation, conidiation, spore germination, and colony growth, compared to the ΔFsBmh1. Notably, both knockout mutants showed a reduction in virulence, with transcriptome analysis revealing changes associated with the observed phenotypes. To further investigate the functional interplay between FsBmh1 and FsBmh2, we constructed and analyzed mutants with combined deletion and silencing (ΔFsBmh/siFsBmh) as well as overexpression (O-FsBmh). The combinations of ΔFsBmh1/siFsBmh2 or ΔFsBmh2/siFsBmh1 displayed more severe phenotypes than those with single allele deletions, suggesting a functional redundancy between the two 14-3-3 proteins. Yeast two-hybrid (Y2H) assays identified 20 proteins with pivotal roles in primary metabolism or diverse biological functions, 12 of which interacted with both FsBmh1 and FsBmh2. Three proteins were specifically associated with FsBmh1, while five interacted exclusively with FsBmh2. In summary, this research provides novel insights into the roles of FsBmh1 and FsBmh2 in F. sacchari and highlights potential targets for PBD management through the modulation of FsBmh functions.

Abstract 1259: HMGA1: An epigenetic gatekeeper of Wnt signals during colon tumorigenesis and regeneration

Abstract Cancer cells undergo epigenetic reprogramming to co-opt stem cell networks and drive tumor progression, although how this occurs remains unclear. The High Mobility Group A1 (HMGA1) gene encodes an architectural transcription factor that binds to DNA at AT-rich sequences where it recruits chromatin complexes to modulate gene expression. HMGA1 is highly expressed during embryogenesis and in adult stem cells, but silenced in differentiated cells. HMGA1 becomes re-expressed in aggressive cancers where high levels portend adverse outcomes. In colorectal cancer (CRC), HMGA1 is among the genes most highly overexpressed compared to normal colon epithelium. We previously discovered that HMGA1 is required for oncogenic phenotypes in CRC cell lines by inducing genes involved in an epithelial-mesenchymal transition. Hmga1 transgenic mice also develop aberrant proliferation and polyposis of small intestinal epithelium where Hmga1 amplifies Wnt signals to enhance self-renewal. To determine how HMGA1 functions in colon epithelial homeostasis and tumorigenesis, we examined mouse models with overexpression or deficiency of Hmga1. Here, we uncover a previously unknown role for HMGA1 as an epigenetic gatekeeper of Wnt signals in colon epithelial cells, both during tumorigenesis driven by mutant APC or stress-induced tissue repair. Strikingly, loss of just a single Hmga1 allele within the colon epithelium disrupts tumorigenesis while prolonging survival in two models of colon tumorigenesis driven by APC mutation, including CDX2P-CreERTCApcfl/fl and APCMin mice inoculated with pro-carcinogenic, enterotoxigenic Bacteroides fragilis. Single cell RNA sequencing in Apc mutant crypt epithelial cells reveals that intact Hmga1 is required for expansion in the Lgr5+ stem cell pool, deep secretory cells, and tuft cells during tumorigenesis. Hmga1 deficiency impacts the stem cell pool the greatest, decreasing Lgr5+ stem cells with skewing to more proliferative, transit amplifying-like cells. Single cell transcriptomic analyses together with assays of single cell chromatin accessibility demonstrate that Hmga1 maintains accessible chromatin to activate gene loci involved in Wnt signals, including Ascl2, a stem cell master regulator, Wnt agonist receptors, and Wnt downstream effectors. Surprisingly, either depletion or overexpression of Hmga1 has no impact on colon epithelial regeneration under homeostatic conditions, although Hmga1 overexpression enhances tissue repair and survival following injury induced by inflammation or irradiation. Together, our results establish HMGA1 as an epigenetic gatekeeper of Wnt signals in colon stem cells during APC-driven tumorigenesis or injury, but not steady state homeostasis, highlighting its role as a promising therapeutic target for both cancer and regenerative medicine. Citation Format: Linda M. S. Resar, Li Z. Luo, Lingling Xian, Iliana Herrera, Shaoguang Wu, Jung-Hyun Kim, Ying Feng, Julian Calderon-Espinosa, Eric R. Fearon, Cynthia Sears. HMGA1: An epigenetic gatekeeper of Wnt signals during colon tumorigenesis and regeneration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1259.

The effect of marker types and density on genomic prediction and GWAS of key performance traits in tetraploid potato.

Genomic prediction and genome-wide association studies are becoming widely employed in potato key performance trait QTL identifications and to support potato breeding using genomic selection. Elite cultivars are tetraploid and highly heterozygous but also share many common ancestors and generation-spanning inbreeding events, resulting from the clonal propagation of potatoes through seed potatoes. Consequentially, many SNP markers are not in a 1:1 relationship with a single allele variant but shared over several alleles that might exert varying effects on a given trait. The impact of such redundant "diluted" predictors on the statistical models underpinning genome-wide association studies (GWAS) and genomic prediction has scarcely been evaluated despite the potential impact on model accuracy and performance. We evaluated the impact of marker location, marker type, and marker density on the genomic prediction and GWAS of five key performance traits in tetraploid potato (chipping quality, dry matter content, length/width ratio, senescence, and yield). A 762-offspring panel of a diallel cross of 18 elite cultivars was genotyped by sequencing, and markers were annotated according to a reference genome. Genomic prediction models (GBLUP) were trained on four marker subsets [non-synonymous (29,553 SNPs), synonymous (31,229), non-coding (32,388), and a combination], and robustness to marker reduction was investigated. Single-marker regression GWAS was performed for each trait and marker subset. The best cross-validated prediction correlation coefficients of 0.54, 0.75, 0.49, 0.35, and 0.28 were obtained for chipping quality, dry matter content, length/width ratio, senescence, and yield, respectively. The trait prediction abilities were similar across all marker types, with only non-synonymous variants improving yield predictive ability by 16%. Marker reduction response did not depend on marker type but rather on trait. Traits with high predictive abilities, e.g., dry matter content, reached a plateau using fewer markers than traits with intermediate-low correlations, such as yield. The predictions were unbiased across all traits, marker types, and all marker densities >100 SNPs. Our results suggest that using non-synonymous variants does not enhance the performance of genomic prediction of most traits. The major known QTLs were identified by GWAS and were reproducible across exonic and whole-genome variant sets for dry matter content, length/width ratio, and senescence. In contrast, minor QTL detection was marker type dependent.

Molecular mechanisms of differentiation and class choice of olfactory sensory neurons.

The sense of smell is intricately linked to essential animal behaviors necessary for individual survival and species preservation. During vertebrate evolution, odorant receptors (ORs), responsible for detecting odor molecules, have evolved to adapt to changing environments, transitioning from aquatic to terrestrial habitats and accommodating increasing complex chemical environments. These evolutionary pressures have given rise to the largest gene family in vertebrate genomes. Vertebrate ORs are phylogenetically divided into two major classes; class I and class II. Class I OR genes, initially identified in fish and frog, have persisted across vertebrate species. On the other hand, class II OR genes are unique to terrestrial animals, accounting for ~90% of mammalian OR genes. In mice, each olfactory sensory neuron (OSN) expresses a single functional allele of a single OR gene from either the class I or class II OR repertoire. This one neuron-one receptor rule is established through two sequential steps: specification of OR class and subsequent exclusive OR expression from the corresponding OR class. Consequently, OSNs acquire diverse neuronal identities during the process of OSN differentiation, enabling animals to detect a wide array of odor molecules. This review provides an overview of the OSN differentiation process through which OSN diversity is achieved, primarily using the mouse as a model animal.

Network-based analysis predicts interacting genetic modifiers from a meta-mapping study of spike-wave discharge in mice.

Absence seizures are characterized by brief lapses in awareness accompanied by a hallmark spike-and-wave discharge (SWD) electroencephalographic pattern and are common to genetic generalized epilepsies (GGEs). While numerous genes have been associated with increased risk, including some Mendelian forms with a single causal allele, most cases of GGE are idiopathic and there are many unknown genetic modifiers of GGE influencing risk and severity. In a previous meta-mapping study, crosses between transgenic C57BL/6 and C3HeB/FeJ strains, each carrying one of three SWD-causing mutations (Gabrg2tm1Spet(R43Q) , Scn8a8j or Gria4spkw1 ), demonstrated an antagonistic epistatic interaction between loci on mouse chromosomes 2 and 7 influencing SWD. These results implicate universal modifiers in the B6 background that mitigate SWD severity through a common pathway, independent of the causal mutation. In this study, we prioritized candidate modifiers in these interacting loci. Our approach integrated human genome-wide association results with gene interaction networks and mouse brain gene expression to prioritize candidate genes and pathways driving variation in SWD outcomes. We considered candidate genes that are functionally associated with human GGE risk genes and genes with evidence for coding or non-coding allele effects between the B6 and C3H backgrounds. Our analyses output a summary ranking of gene pairs, one gene from each locus, as candidates for explaining the epistatic interaction. Our top-ranking gene pairs implicate microtubule function, cytoskeletal stability and cell cycle regulation as novel hypotheses about the source of SWD variation across strain backgrounds, which could clarify underlying mechanisms driving differences in GGE severity in humans.

Mineralocorticoid receptor and aldosterone: Interaction between NR3C2 genetic variants, sex and age in a mixed cohort.

Hypertension, a prevalent cardiovascular risk, often involves dysregulated aldosterone and its interaction with the mineralocorticoid receptor (MR). Experimental designs in animal models and human cohorts have demonstrated a sex and age dependency of aldosterone secretion that expands our pathophysiologic understanding. This study explores the genetic variation of NR3C2, which encodes MR, in relation to aldosterone, considering age, sex, and race. Incorporating 720 Caucasians and 145 Africans from the HyperPATH cohort, we investigated the impact of rs4835490, a single nucleotide risk allele variant, on aldosterone levels and vasculature. Notably, a significant association between rs4835490 and plasma aldosterone under liberal salt conditions emerged in individuals of European ancestry (P=0.0002). Homozygous carriers of the risk A allele exhibited elevated plasma aldosterone levels (AA=8.1±0.9 vs GG=4.9±0.5 ng/dl). Additionally, aldosterone activation through posture (P=0.025) and urinary excretion (P=0.0122) showed notable associations. Moreover, genetic interactions with race, sex, and age were observed. Caucasian females under 50 years displayed higher plasma aldosterone, urine aldosterone, and posture aldosterone with the AA genotype compared to females over 50 years, suggesting a potential connection with menopausal or estrogen influences. Interestingly, such age-dependent interactions were absent in the African cohort. our study highlights the significance of NR3C2 genetic variation and its interplay with age, sex, and race in aldosterone activation. The findings point towards an estrogen-modulating effect on MR activation, particularly in women underlining the role of aldosterone dysregulation in hypertension development. This insight advances our comprehension of hypertension's complexities and opens avenues for personalized interventions.

Genetic mapping reveals new loci and alleles for flowering time and plant height using the double round-robin population of barley.

Flowering time and plant height are two critical determinants of yield potential in barley (Hordeum vulgare). Despite their role in plant physiological regulation, a complete overview of the genetic complexity of flowering time and plant height regulation in barley is still lacking. Using a double round-robin population originated from the crossings of 23 diverse parental inbred lines, we aimed to determine the variance components in the regulation of flowering time and plant height in barley as well as to identify new genetic variants by single and multi-population QTL analyses and allele mining. Despite similar genotypic variance, we observed higher environmental variance components for plant height than flowering time. Furthermore, we detected new QTLs for flowering time and plant height. Finally, we identified a new functional allelic variant of the main regulatory gene Ppd-H1. Our results show that the genetic architecture of flowering time and plant height might be more complex than reported earlier and that a number of undetected, small effect, or low-frequency genetic variants underlie the control of these two traits.

Cytochrome P450 Family 4F2 and 4F11 Haplotype Mapping and Association with Hepatic Gene Expression and Vitamin K Hydroxylation Activity.

This study evaluated the underlying mechanistic links between genetic variability in vitamin K metabolic pathway genes (CYP4F2 and CYP4F11) and phylloquinone hydroxylation activity using genotype- and haplotype-based approaches. Specifically, we characterized genetic variability in the CYP4F2/CYP4F11 locus and compared common single allele genotypes and common haplotypes as predictors of hepatic gene expression, enzyme abundance, and phylloquinone (VK1) ω-hydroxylation kinetics. We measured CYP4F2 and CYP4F11 mRNA levels, CYP4F2 and CYP4F11 protein abundances, and the VK1 concentration-dependent ω-hydroxylation rate in matched human liver nucleic acid and microsome samples, utilizing a novel in vitro population modeling approach. Results indicate that accounting for the CYP4F2*3 allele alone is sufficient to capture most of the genetic-derived variability in the observed phenotypes. Additionally, our findings highlight the important contribution that CYP4F11 makes toward vitamin K metabolism in the human liver.

STAT6 tunes maximum T cell IL-4 production from stochastically regulated Il4 alleles.

T helper 2 (Th2) cells stochastically express from the Il4 locus but it has not been determined whether allelic expression is linked or independent. Here, we provide evidence that alleles are independently activated and inactivated. We compared Il4 locus expression in T cells from hemizygous IL-4 reporter mice in culture and in vivo following exposure to type 2 immunogens. In culture, Il4 alleles had independent, heritable expression probabilities. Modeling showed that in co-expressors, dual allele transcription occurs for only short periods, limiting per-cell mRNA variation in individual cells within a population of Th2 cells. In vivo profiles suggested that early in the immune response, IL-4 output was derived predominantly from single alleles, but co-expression became more frequent over time and were tuned by STAT6, supporting the probabilistic regulation of Il4 alleles in vivo among committed IL-4 producers. We suggest an imprinted probability of expression from individual alleles with a short transcriptional shutoff time controls the magnitude of T cell IL-4 output, but the amount produced per allele is amplified by STAT6 signaling. This form of regulation may be a relevant general mechanism governing cytokine expression.

Ku70 senses cytosolic DNA and assembles a tumor-suppressive signalosome.

The innate immune response contributes to the development or attenuation of acute and chronic diseases, including cancer. Microbial DNA and mislocalized DNA from damaged host cells can activate different host responses that shape disease outcomes. Here, we show that mice and humans lacking a single allele of the DNA repair protein Ku70 had increased susceptibility to the development of intestinal cancer. Mechanistically, Ku70 translocates from the nucleus into the cytoplasm where it binds to cytosolic DNA and interacts with the GTPase Ras and the kinase Raf, forming a tripartite protein complex and docking at Rab5+Rab7+ early-late endosomes. This Ku70-Ras-Raf signalosome activates the MEK-ERK pathways, leading to impaired activation of cell cycle proteins Cdc25A and CDK1, reducing cell proliferation and tumorigenesis. We also identified the domains of Ku70, Ras, and Raf involved in activating the Ku70 signaling pathway. Therapeutics targeting components of the Ku70 signalosome could improve the treatment outcomes in cancer.

Advances in the Study of Common and Rare CFTR Complex Alleles Using Intestinal Organoids.

Complex alleles (CAs) arise when two or more nucleotide variants are present on a single allele. CAs of the CFTR gene complicate the cystic fibrosis diagnosis process, classification of pathogenic variants, and determination of the clinical picture of the disease and increase the need for additional studies to determine their pathogenicity and modulatory effect in response to targeted therapy. For several different populations around the world, characteristic CAs of the CFTR gene have been discovered, although in general the prevalence and pathogenicity of CAs have not been sufficiently studied. This review presents examples of using intestinal organoid models for assessments of the two most common and two rare CFTR CAs in individuals with cystic fibrosis in Russia.

Identification of Fusarium wilt resistance gene SiRLK1 in Sesamum indicum L.

Sesame Fusarium wilt (SFW), caused by Fusarium oxysporum f. sp. sesami (Fos), is one of the most devastating diseases affecting sesame cultivation. Deciphering the genetic control of SFW resistance is pivotal for effective disease management in sesame. An inheritance study on a cross between the highly resistant variety Yuzhi 11 and the highly susceptible accession Sp1 using a Fos pathogenicity group 1 isolate indicated that resistance was conferred by a single dominant allele. The target locus was located in a 1.24 Mb interval on chromosome 3 using a combination of cross-population association mapping and bulked segregant analysis. Fine genetic mapping further narrowed the interval between 21,350 and 21,401 kb. The locus Sindi_0812400 was identified as the SFW resistance gene and officially designated SiRLK1. This gene encodes a specific malectin/receptor-like protein kinase with three putative tandem kinase domains and is considered a kinase fusion protein. Sequence analysis revealed that a high proportion (49.44%) of variants within the locus was located within the kinase domain III, and several of which were evidently associated with the diversity in SFW response, indicating the critical role of kinase domain III in expression of disease resistance. These findings provide valuable information for further functional analysis of SFW resistance genes and marker-assisted resistance breeding in sesame.