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At least 19 recordsLinked to original sources
PubMed2026

Epi-Allele elicits compensatory expression of the non-targeted allele and prevents haploinsufficiency in dominant genetic diseases.

Epigenetic regulation may underlie asymmetric allelic expression of many genes during development and disease pathogenesis. Allele-specific epigenetic modification could provide an efficient therapy for dominant genetic diseases due to heterozygous mutations. We developed an allele-specific epigenetic editing method ("Epi-Allele") for silencing pathogenic alleles and found surprisingly elevated expression of the non-targeted alleles, leaving total gene expression unchanged. Genome-wide screening revealed that such compensated allelic expression represents a common phenomenon, suggesting that the Epi-Allele approach could avoid the haploinsufficiency induced by current allele-specific silencing therapies. This notion was validated by allele-specific epigenetic remodeling of Myh6 and MYH7 genes in ameliorating cardiac phenotypes in a hypertrophic cardiomyopathy (HCM) mouse model and HCM patient iPSC-derived cardiomyocytes, respectively. Thus, Epi-Allele offers an allele-specific haploinsufficiency-free therapeutic approach for treating dominant genetic diseases.

DNA methylation↗
PubMed2026

Polysomal Profiling Coupled to Allele-Specific Proteomics Reveals an EIF4H TranSNP Allele Possessing Higher mRNA Translation Potential.

To search for genetic sources of allele-specific mRNA translation, we leveraged heterozygous polymorphisms and variants present in the exome of HCT116 colorectal adenocarcinoma-derived cells, computing allelic fractions from both total and polysome-associated RNA from RNA-Seq data. Allelic imbalance in polysomal RNA led us to nominate 52 coding variants associated with allele-specific mRNA translation, of which 16 are nonsynonymous. To validate instances of allele-specific translation, a proteomics workflow was developed that combines label-free shotgun analysis, high-pH reversed-phase peptide fractionation, and targeted parallel reaction monitoring using isotope-labeled peptide standards. Using this approach, we provide proof-of-concept validation of the heterozygous G>A, R183H missense single-nucleotide variant rs1554710467 in the eukaryotic initiation factor 4H (EIF4H) gene. The variant is present in two EIF4H alternatively spliced variants, which showed equivalent translation efficiency in HCT116 cells but differ in abundance. The alternative peptide containing H183 was significantly more abundant than the corresponding reference peptide containing R183, consistent with the over-representation of the alternative allele in polysomal RNA in HCT116 cells. A dual-fluorescence ribosome-stalling assay confirmed the enhanced translation potential of the variant allele. The two EIF4H allelic proteins exhibited similar stability and subpolysomal localization. This study demonstrates the feasibility of using allele-specific proteomics at the endogenous protein levels by exploiting heterozygous coding variants. Overall, our approach extends the toolbox available to investigate allele-specific differences in mRNA translation potential, a relatively underexplored layer of gene expression regulation that could reveal interindividual differences in disease-relevant phenotypes.

Humans↗
PubMed1976

Heterozygous familial hypercholesterolemia: failure of normal allele to compensate for mutant allele at a regulated genetic locus.

In normal human fibroblasts, the synthesis of a cell surface receptor for plasma low density lipoprotein (LDL) is regulated by a sensitive system of feedback suppression. The number of functional LDL receptors declines by more than 20 fold when cellular stores of esterified cholesterol are increased by incubation of cells with an exogenous source of cholesterol. Fibroblasts from patients with the heterozygous form of familial hypercholesterolemia (FH) possess one functional allele and one nonfunctional allele at the LDL receptor locus. In the current studies, we have examined the effect that this deficiency produces upon the pattern of regulation of the single functional allele at the LDL receptor locus. Under growth conditions that induced a maximal rate of LDL receptor synthesis (that is, growth in the absence of an exogenous source of cholesterol), the FH heterozygote cells produced about one half as many functional LDL receptors as did the normal cells. More importantly, when grown in the presence of increasing amounts of exogenous cholesterol, the FH heterozygote and normal cells suppressed their respective LDL receptor activities in parallel. Over a wide range of LDL receptor activities, at each level of cellular esterified cholesterol, the FH heterozygote cells expressed about one half as many receptors as did the normal cells. These data indicate that in the FH heterozygote cells, the receptor regulatory mechanism dictates that the normal allele produce only the amount of gene product that it would normally produce at a given level of cellular esterified cholesterol. The failure of the regulatory mechanism to stimulate the normal allele at the LDL receptor locus to produce twice its normal amount of gene product leaves the FH heterozygote cells with a persistent 50% deficiency in LDL receptors under all conditions of cell growth.

Alleles↗
PubMed1978

Allelic expression in intergeneric fox hybrids (Alopex lagopus x Vulpes vulpes). III. Regulation of the expression of the parental alleles at the Gpd locus linked to the X chromosome.

The electrophoretic pattern of glucose-6-phosphate dehydrogenase (G6PD) was studied in 60 intergeneric fox hybrids (Alopex lagopus x Vulpes vulpes), 33 females and 27 males. It is shown that the structural gene for G6PD, designated Gpd, is located on the X chromosome in both Arctic and silver foxes. Analysis of G6PD patterns in the erythrocytes of hybrid females demonstrated that the phenotypic expression of parental alleles at the Gpd locus varied considerably: from 1:1 to the hemizygous manifestation of an allele of either the Artic or the silver fox. The expression of the parental alleles at this locus is different in the various tissues of single female hybrids. It is suggested that the variable quantitative expression of the alleles at the Gpd locus in hybrid females is related to the presence of two cell populations having in an active state either the X chromosome of the Arctic fox or that of the silver fox. It is also proposed that the size of the two cell populations is largely affected by the different relationships between cells having different activated X-chromosomes among initiator (stem) cells from which various definitive organs and tissues develop. The number of initiator cells for erythroid tissue has been calculated to be five or six.

Alleles↗
PubMed1975

Allelic negative complementation at the Abruptex locus of Drosophila melanogaster.

The mutations of the Abruptex locus in Drosophila melanogaster fall into three categories. There are recessive lethal alleles and viable alleles. The latter can be divided into suppressors and nonsuppressors of Notch mutations. The recessive lethals are lethal in heterozygous combination with Notch. As a rule the recessive lethals are lethal also in heterozygous combination with the viable alleles. Heterozygous combinations of certain viable alleles are also lethal. In such heterozygotes, one heteroallele is a suppressor of Notch and the other is a nonsuppressor. Other heterozygous combinations of viable alleles are viable and have an Abruptex phenotype. The insertion of the wild allele of the Abruptex locus as an extra dose (carried by a duplication) into the chromosomal complement of the fly fully restores the viability of the otherwise lethal heterozygotes if two viable alleles are involved. The extra wild allele also restores the viability of heterozygotes in which a lethal and a suppressor allele are present. If, however, a lethal and a nonsuppressor are involved, the wild allele only partly restores the viability, and the effect of the wild allele is weakest if two lethal alleles are involved. It seems likely that of the viable alleles the suppressors of Notch are hypermorphic and the nonsuppressors are hypomorphic. The lethal alleles share properties of both types, and are possibly antimorphic mutations. It is suggested that the locus is responsible for a single function which, however, consists of two components. The hypermorphic mutations are defects of the one component and the hypomorphic mutations of the other. In heterozygotes their cumulative action leads to decreased viability. The lethal alleles are supposed to be defects of the function as a whole. The function controlled by the locus might be a regulative function.

Alleles↗
PubMed2025

Carrying APOL1 G1 allele is associated with cardiovascular complications during COVID-19 in an admixed population.

BACKGROUND: The APOL1 G1 and G2 alleles were selected in the Sub-Saharan African population by conferring resistance to trypanosome infection. However, these alleles are associated with kidney diseases, and their role in cardiovascular complications remains uncertain. A second hit mediated by an inflammatory state is necessary for APOL1-mediated phenotypes. Thus, this cross-sectional study investigates the association of APOL1 alleles with COVID-19 outcomes such as cardiovascular complications and kidney injury in an admixed population. Whole-genome sequencing was performed for 485 patients with different outcomes from a Biobank in Southern Brazil. RESULTS: COVID-19 individuals presented median age of 51 years, 281 were hospitalized, and 10.9% had CKD previous to the infection. Global ancestry inference revealed 12.8% of African ancestry. The G1 allele frequency was 2.7% and G2 allele was 1.2%. Local ancestry inference evidenced African ancestry in the locus of APOL1 alleles. The G1 allele frequency was higher among patients with severe outcomes. The presence of this allele was associated with kidney injury (OR = 2.78; 95% CI = 1.04-7.42; p = 0.041) using a minimally adjusted model and cardiovascular complications with a minimally (OR = 4.61; 95% CI = 1.61-13.19; p = 0.004) and fully adjusted model (OR = 4.59; 95% CI = 1.41-14.96; p = 0.011). Four individuals carried two alleles (three G1/G1 and one G1/G2) and three of them progressed to severe COVID-19 developing kidney injury. CONCLUSION: APOL1 risk alleles are present in the Brazilian population due to genetic admixture and the G1 allele was associated with COVID-19 outcomes.

Humans↗
PubMed2026

Clinical Function Assignment of NAT2 Alleles by the Clinical Pharmacogenetics Implementation Consortium Pharmacogene Curation Expert Panel.

NAT2 encodes arylamine N-acetyltransferase 2, a key enzyme in the phase II metabolism of arylamines and arylhydrazines. NAT2 is highly polymorphic, resulting in variable distributions of rapid and poor metabolizers across global populations. Here, we detail the process undertaken by the Clinical Pharmacogenetics Implementation Consortium (CPIC) NAT2 Pharmacogene Curation Expert Panel (PCEP) to assign clinical function to NAT2 star (*) alleles using CPIC's standard terminology. Given the observed impact of NAT2 genetic variability on drug response, CPIC convened the NAT2-PCEP to standardize clinical allele function assignments. The NAT2-PCEP is comprised of multidisciplinary and international members, including researchers, clinicians, and implementers with expertise in pharmacogenomics and NAT2 molecular biology. Extensive in vitro and clinical literature was curated from PubMed and other sources to assess NAT2 genotype-to-phenotype concordance as well as the biochemical function of NAT2 star alleles. The NAT2-PCEP assigned allele clinical function using CPIC's standard terminology (increased, decreased, uncertain, and unknown function) to 59 star alleles cataloged by the Pharmacogene Variation Consortium (PharmVar). Two alleles, NAT2*1 and NAT2*4, were assigned increased function (historically known as rapid), 40 alleles were assigned decreased function (historically known as slow), 10 alleles were assigned uncertain function, and seven alleles were assigned unknown function. Rigorous evidence review and in-depth PCEP discussion were crucial in determining these function assignments. The findings reported here underscore the importance of standardized allele functional terms and diplotype-to-phenotype assignments to further the clinical implementation of NAT2 pharmacogenetic test results.

Arylamine N-Acetyltransferase↗
PubMed2026

Innovative CRISPR/Cas9-Based Strategy for Allele-Specific HLA Peptidome Analysis Using a Pan-HLA Antibody.

Human leukocyte antigen (HLA) immunopeptidomics is restricted by the limited availability of allele-specific antibodies and by potential artifacts introduced by HLA overexpression systems. To address these challenges, we developed a CRISPR/Cas9-based strategy that selectively deletes undesired classical class I alleles while preserving a single endogenous allele, thereby enabling allele-resolved peptidome profiling with a pan-HLA class I antibody. As a proof of concept, we edited JY cells to eliminate HLA-B∗07:02 and HLA-C∗07:02 while retaining HLA-A∗02:01 (ΔBC clones). Peptide-HLA complexes were immunoprecipitated from WT and ΔBC clones using either the pan-HLA class I antibody W6/32 or the A∗02:01-specific antibody PA2.1, followed by nanoLC-MS/MS and computational HLA assignment. Deletion of HLA-B and HLA-C alleles caused an expected ∼55% reduction in total class I surface expression. Despite this, W6/32 immunoprecipitation from ΔBC clones recovered a comparable peptide yield to PA2.1 in WT cells. Binding predictions showed that most peptides identified in ΔBC clones using W6/32 were assigned to HLA-A∗02:01, with near-complete loss of HLA-B∗07:02- and HLA-C∗07:02-derived peptides. Sequence logo analysis confirmed the canonical A∗02:01 motif across conditions. The ΔBC W6/32 immunopeptidome exhibited a high degree of overlap (∼88%) with the WT PA2.1 repertoire, supporting the specificity and fidelity of the approach. These findings establish CRISPR-based editing of HLA alleles as a viable strategy for allele-specific immunopeptidome analysis using pan-HLA antibodies, supporting its potential application beyond this proof-of-concept system, reducing reliance on allele-specific reagents and facilitating the study of underrepresented HLA alleles.

Humans↗
PubMed1979

Switching of a mating-type a mutant allele in budding yeast Saccharomyces cerevisiae.

Aimed at investigating the recovery of a specific mutant allele of the mating type locus (MAT) by switching a defective MAT allele, these experiments provide information bearing on several models proposed for MAT interconversion in bakers yeast, Saccharomyces cerevisiae. Hybrids between heterothallic (ho) cells carrying a mutant MAT a allele, designated mata-2, and MAT alpha ho strains show a high capacity for mating with MATa strains. The MAT alpha/mata-2 diploids do not sporulate. However, zygotic clones obtained by mating MAT alpha homothallic (HO) cells with mata-2 ho cells are unable to mate and can sporulate. Tetrad analysis of such clones revealed two diploid (MAT alpha/MATa):two haploid segregants. Therefore, MAT switches occur in MAT alpha/mata-2 HO/ho cells to produce MAT alpha/Mata cells capable of sporulation. In heterothallic strains, the mata-2 allele can be switched to a functional MAT alpha and subsequently to a functional MATa. Among 32 MAT alpha to MATa switches tested, where the MAT alpha was previously derived from the mata-2 mutant, only one mata-2 like isolate was observed. However, the recovered allele, unlike the parental allele, complements the matalpha ste1-5 mutant, suggesting that these alleles are not identical and that the recovered allele presumably arose as a mutation of the Mat alpha locus. No mata-2 was recovered by HO-mediated switching of MAT alpha (previously obtained from mata-2 by HO) in 217 switches analyzed. We conclude that in homothallic and heterothallic strains, the mata-2 allele can be readily switched to a functional MAT alpha and subsequently to a functional MATa locus. Overall, the results are in accord with the cassette model (HICKS, STRATHERN and HERSKOWITZ )977b) proposed to explain MAT interconversions.

Alleles↗
PubMed2026

The 2 breakpoint regions of an RHCE-D(2-9)-CE allele causing a D phenotype and its RhAG antigen density.

BACKGROUND: Among 195 known RHCE alleles, 10 have evolved to form hybrid RHCE-D-CE alleles encoding aberrant RhCE proteins lacking CE antigen expression. The resulting D-- phenotype predisposes to anti-Hro alloimmunization (anti-Rh17). Although hybrid alleles may appear identical at the mRNA level, distinct breakpoint configurations can result in diverse D phenotypes or Rh antigens. We determined the molecular structure of a D-- phenotype. An unrelated RHD allele, DMA had been observed once without serology. We tested Rh antigen densities. MATERIALS AND METHODS: Genomic DNA and cDNA were analyzed using a combination of molecular techniques and commercial red cell genotyping assays targeting the RHD and RHCE genes. A flow cytometric method was developed to quantify RhAG antigen expression. Red cell antigen densities were determined for RhAG and RhD in the D-- and DMA/DAU3 samples serologically. RESULTS: Nucleotide sequencing of the RHCE gene and its cDNA identified a homozygous CE-D(2-9)-CE hybrid allele in an individual of Afghan origin. The 5' and 3' breakpoint regions included 131 and 4,287 nucleotides. The new allele was linked in a haplotype to the normal RHD allele (RHD*01). Quantitative flow cytometry demonstrated antigen densities of 92,297 RhD and 52,907 RhAG molecules per red cell for the D-- sample (12,465 RhD and 117,871 RhAG for DMA/DAU3). DISCUSSION: We describe the breakpoint regions of a Ce-D(2-9)-Ce allele that encoded a D-- phenotype. Our results underscore the importance of breakpoint characterization for identifying clinically relevant RH variants and advancing personalized transfusion medicine. The second DMA observation, in trans to a DAU3 allele, aligned with a weak D phenotype for DMA.

Journal Article↗
PubMed2025

Development of PCR-based markers for the identification of wheat HMW glutenin subunit alleles at the GLU-A1 and GLU-D1 loci.

The allelic variations of high-molecular-weight glutenin subunit locus in common wheat (Triticum aestivum L.) markedly influence grain end-use quality. GLU-A1, GLU-B1, and GLU-D1, which encode high-molecular-weight glutenin subunits, are located on the long arms of chromosomes 1A, 1B, and 1D, respectively. However, existing markers for distinguishing alleles at the GLU-A1 and GLU-D1 are limited with regard to both number and resolution. In the present study, we enhanced the utility of PCR-based allele detection by developing seven new agarose gel-based markers capable of differentiating four Glu-A1x, four Glu-D1x, and two Glu-D1y alleles. These new markers, in combination with previously published PCR markers, were used to successfully identify the Glu-A1 × 1, Glu-A1 x 2*, Glu-A1 x 2.1*, and Glu-A1x-null alleles and the Glu-D1 x 5, Glu-D1 x 2, Glu-D1 x 2.1, and Glu-D1 x 2.2 alleles across 25 wheat resources. Additionally, we developed a novel marker that enables us to distinguish between the Glu-D1y10 and Glu-D1y12 alleles more clearly than conventional markers. These improved PCR markers represent a reliable and efficient tool for detecting allelic variations at the GLU-A1 and GLU-D1 loci. They are expected to serve as valuable resources for marker-assisted selection and marker-assisted backcrossing aimed at improving the processing quality of wheat.

Triticum↗
PubMed2025

Polaris: Polarization of ancestral and derived polymorphic alleles for inferences of extended haplotype homozygosity in human populations.

SUMMARY: Statistical methods that measure the extent of haplotype homozygosity on chromosomes have been highly informative for identifying episodes of recent selection. For example, the integrated haplotype score (iHS) and the extended haplotype homozygosity (EHH) statistics detect long-range haplotype structure around derived and ancestral alleles indicative of classic and soft selective sweeps, respectively. However, to our knowledge, there are currently no publicly available methods that classify ancestral and derived alleles in genomic datasets for the purpose of quantifying the extent of haplotype homozygosity. Here, we introduce the Polaris package, which polarizes chromosomal variants into ancestral and derived alleles and creates corresponding genetic maps for analysis by selscan and HaploSweep, two versatile haplotype-based programs that perform scans for selection. With the input files generated by Polaris, selscan and/or HaploSweep can produce the appropriate sign (either positive or negative) for outlier iHS statistics, enabling users to distinguish between selection on derived or ancestral alleles. In addition, Polaris can convert the numerical output of these analyses into graphical representations of selective sweeps, increasing the functionality of our software. RESULTS: To demonstrate the utility of our approach, we applied the Polaris package to Chromosome 2 in the European Finnish, Middle Eastern Bedouin, and East African Maasai populations. More specifically, we examined the regulatory sequence in intron 13 of the MCM6 gene associated with lactase persistence (i.e. the ability to digest the lactose sugar present in fresh milk), a region of intense interest to human evolutionary geneticists. Our analyses showed that derived alleles (at known enhancers for lactase expression) sit on an extended haplotype background in the Finnish, Bedouin, and Maasai consistent with a classic selective sweep model as determined by iHS and EHH statistics. Importantly, we were able to immediately identify this target allele under selection based on the information generated by our software. We also explored outlier statistics across Chromosome 2 in two distinct datasets from these populations: (i) one containing polarized alleles generated with Polaris and (ii) the other containing unpolarized alleles in the original phased vcf file. Here, we found an excess of outlier statistics on Chromosome 2 in the unpolarized datasets, raising the possibility that a subset of these "hits" of selection may be unreliable. Overall, Polaris is a versatile package that enables users to efficiently explore, interpret, and report signals of recent selection in genomic datasets. AVAILABILITY AND IMPLEMENTATION: The Polaris package is free and open source on GitHub (https://github.com/alisi1989/Polaris) and DropBox (https://www.dropbox.com/scl/fo/mlxizft5267vem9u62qkn/AAnM0qX923zPzQBlPX8iteM?rlkey=uezrp4t2waffpj0nmo1evr320&e=1&st=jaodccws&dl=0).

Haplotypes↗
PubMed2026

A de novo algorithm for allele reconstruction from Oxford nanopore amplicon reads, with application to CYP2D6.

MOTIVATION: The Oxford Nanopore Technologies' sequencing platform offers a path towards bedside genomics, producing long reads that can completely cover a gene of interest, and detect any known or novel variant the gene contains. However, the analysis of these long reads to identify actionable genotypes remains challenging and typically requires customization depending on the target gene. RESULTS: Here, we describe a generic algorithm to accurately reconstruct allele sequences derived from long-reads of amplicon-based data. Rather than calling variants directly from these long-reads, our method takes a "sequence-first" approach, performing an unbiased reconstruction of the underlying amplicon sequences to generate high-confidence reconstructed allele sequences. This is done without user input of the target gene, allowing for any source amplicon to be reconstructed. These high-confidence reconstructed allele sequences are then compared to the genomic reference sequence of the gene to infer the specific diplotype present in the sample. This approach is agnostic towards the number of genes and alleles present and readily detects novel variants. We demonstrate our approach using three independent data sets for CYP2D6, a diverse and complex gene with over 175 known alleles of clinical significance. We show how our approach can accurately recover validated CYP2D6 diplotypes from 20 Coriell samples covering 14 distinct alleles, using different amplicons, flow cell versions, and depths. This includes inferring occurrences of allele duplication events from relative abundances of each allele, a critical factor for ascribing functional effects to a diplotype. Further, we demonstrate our approach's utility for other genomic regions, including HLA. AVAILABILITY: Custom code is available at the following GitHub repository, along with instructions for use and test data: https://github.com/scottdbrown/allele-reconstruction-long-read-amplicon-data. A snapshot of the code at the time of publication is available on Zenodo.org; doi 10.5281/zenodo.19716004. Raw .fastq sequence data for our three sequencing runs is available at the SRA under Bioproject PRJNA1357883 (https://www.ncbi.nlm.nih.gov/bioproject/1357883).

Alleles↗
PubMed2026

Haplotype-resolved 3D genome maps reveal RNAPII-mediated allelic regulation in hybrid rice.

To understand how the two parental genomes coordinate transcription in hybrids, chromatin architecture must be resolved at the haplotype level. Here, using phased Bridge-Linker Hi-C, we reconstructed a haplotype-resolved three-dimensional (3D) genome of the elite hybrid rice (Oryza sativa) line Shanyou 63 (SY63). We identified extensive allele-specific chromatin conformations. Furthermore, we generated allele-resolved RNAPII ChIA-PET maps and phased transcriptomes to explore how chromatin interactions contribute to allelic regulation. Although maternal and paternal homologs share broadly similar chromatin features, we detected widespread haplotype-biased RNAPII binding and chromatin looping at high resolution. These allele-specific RNAPII-mediated contacts were significantly associated with biased expression. Stronger RNAPII binding on one haplotype promoted the formation of long-range regulatory loops with distal genes, thereby contributing to allele-biased transcription at a subset of loci, even when promoter-proximal RNAPII occupancy was comparable between alleles. These results demonstrate that subtle differences in RNAPII engagement and 3D regulatory wiring between parental haplotypes can reshape transcriptional output in hybrids, providing new insights into the mechanisms underlying the allelic regulation of gene expression.

Allele-specific chromatin interactions↗
PubMed2026

Immunopeptidomics-driven MHC class II peptide-binding motif discovery for 2 common canine DR alleles.

Despite the central role of major histocompatibility complex (MHC) class II in adaptive immunity, peptide-binding motifs have yet to be characterized for any canine MHC class II allele. Here, we report the first immunopeptidomics-derived binding motifs for DLA-DRB1*015:01 (DLA-DR15) and DLA-DRB1*012:01 (DLA-DR12), 2 alleles overrepresented in breeds predisposed to immune-mediated diseases. Because dogs co-express DLA-DR and DLA-DQ, the MHC class II Ab clone YKIX334.2 was validated to be DLA-DR-specific, enabling allele-selective immunoaffinity purification of DLA-DR molecules from homozygous DLA-DR15 and DLA-DR12 donor spleens. Mass spectrometry and GibbsCluster motif deconvolution of 838 DLA-DR15-associated and 644 DLA-DR12-associated peptides eluted from their respective peptide-binding grooves revealed distinct allele-specific binding motifs, with characterization of anchor residue preferences, peptide-length distributions, cross-species comparisons with human and murine MHC class II motifs, and source protein composition of the eluted self-peptidome. To evaluate the translational utility of these motifs, recombinant DLA-DR15 and DLA-DR12 molecules were used to screen rabies virus glycoprotein and nucleoprotein peptide libraries via fluorescence-based peptide competition assays, identifying high-affinity candidate binders for both alleles. Spearman rank correlation between immunopeptidomics-derived position-specific scoring matrix scores and peptide competition assay rankings demonstrated modest associations, consistent with these approaches capturing complementary dimensions of peptide-MHC class II interaction. Ultimately, these findings establish what we believe is the first allele-specific peptide-binding motif framework for canine MHC class II, providing a foundation for DLA-allele-informed CD4+ T-cell epitope discovery studies and Ag-specific immune response characterization in the dog.

Animals↗
PubMed2026

Sperm subpopulations differing in mitochondrial abundance show divergent nuclear allele frequencies.

Mammalian ejaculates contain heterogeneous sperm subpopulations that differ in subcellular architecture and developmental history, despite appearing morphologically uniform. The extent to which this cellular heterogeneity reflects underlying nuclear genomic structure within a sire remains largely unexplored. Mitochondrial architecture in sperm is established during spermatogenesis, with final assembly and organization occurring during spermiogenesis under nuclear genomic control, positioning variation in mitochondrial abundance and organization as a potential phenomic indicator of within-sire allelic segregation. Here, we tested whether sperm subpopulations defined by differing mitochondrial abundance exhibit systematic differences in nuclear allele representation. Boar sperm were resolved into low and high mitochondrial subpopulations using fluorescence-activated cell sorting based on MitoTracker™ Green fluorescence while excluding debris, doublets, and non-viable cells. Epifluorescence microscopy confirmed that high MitoTracker™ Green fluorescence sperm possessed longer mitochondrial sheaths, validating a structural distinction between subpopulations. Whole-genome sequencing of paired mitochondrial subpopulations from three boars was performed, and allelic ratio distortion was evaluated relative to heterozygous baseline populations. Analyses across heterozygous loci genome-wide identified candidate allele frequency shifts between mitochondrial-defined subpopulations, suggesting non-random segregation of alleles within ejaculates. Using a minimum sequencing depth of 30 reads in both sorted fractions, 182 candidate SNPs were identified with evidence of allele-frequency differences between mitochondrial fluorescence-defined subpopulations. These findings suggest that sperm mitochondrial abundance can potentially serve as an indirect, high-throughput marker of nuclear genomic heterogeneity within sires. This proof-of-concept framework establishes a foundation for future studies integrating sperm phenotyping, genome-wide allele-frequency analysis and functional validation to better characterize gamete-level heterogeneity.

Male↗