Allele-specific Expression Research Articles

GATA2 mutated allele specific expression is associated with a hyporesponsive state of HSC in GATA2 deficiency syndrome

GATA2 germline mutations lead to a syndrome characterized by immunodeficiency, vascular disorders and myeloid malignancies. To elucidate how these mutations affect hematopoietic homeostasis, we created a knock-in mouse model expressing the recurrent Gata2 R396Q missense mutation. Employing molecular and functional approaches, we investigated the mutation’s impact on hematopoiesis, revealing significant alterations in the hematopoietic stem and progenitor (HSPC) compartment in young age. These include increased LT-HSC numbers, reduced self-renewal potential, and impaired response to acute inflammatory stimuli. The mature HSPC compartment was primarily affected at the CMP sub-population level. In the mutant LT-HSC population, we identified an aberrant subpopulation strongly expressing CD150, resembling aging, but occurring prematurely. This population showed hyporesponsiveness, accumulated over time, and exhibited allele-specific expression (ASE) favoring the mutated Gata2 allele, also observed in GATA2 mutated patients. Our findings reveal the detrimental impact of a Gata2 recurrent missense mutation on the HSC compartment contributing to its functional decline. Defects in the CMP mature compartment, along with the inflammatory molecular signature, explain the loss of heterogeneity in HPC compartment observed in patients. Finally, our study provides a valuable model that recapitulates the ASE-related pathology observed in GATA2 deficiency, shedding light on the mechanisms contributing to the disease’s natural progression.

ScRNA seq of an F1 cross of Marek’s disease resistant and susceptible chickens identifies allele specific expression signatures enriched in transcription modulators

Marek’s disease (MD), a T cell lymphoma disease in chickens, is caused by the Marek’s disease virus (MDV) found ubiquitously in the poultry industry. Genetically resistant Line 63 (L6) and susceptible Line 72 (L7) chickens have been instrumental to research on avian immune system response to MDV infection. In this study we characterized molecular signatures unique to splenic immune cell types across different genetic backgrounds 6 days after infection. Using three populations, L6, L7, and an F1 cross between L6xL7, we evaluated the immune cell transcriptome of responding cell types using single cell RNA sequencing. Several MDV genes were found expressed mainly in cytotoxic T cells while ICP4 and MEQ MDV genes were expressed across infected cell types. Using the F1 we quantified allele specific expression (ASE) of biallelic SNPs and found biased expression of parental alleles specific to immune cell subtypes. We identified 22 SNPs with ASE in response to MDV infection mapped to gene rich regions surrounding 59 genes of critical importance for chromatin remodeling and transcriptional regulation. Histone deacetylase genes (HDAC1 and HDAC8) had increased expression of L6 alleles, while small nuclear RNA genes (SNORA68 and SNORA72) expressed higher levels of L7 alleles with infection in T cell subsets. SNPs with ASE also mapped genes important for an adequate immune response including GNLY (cytotoxic activity) and PDIA3 (component of MHC class I peptide loading complex), and genes known to promote viral replication (MCM5 and EIF3M). These results show that functional variants associated with susceptibility to MD may have a bigger impact in subsets of immune cell types, and by characterizing the transcriptomes of these subtypes we can unravel molecular signatures specific to MD genomic resistance.

Significance of KLK7 expression, polymorphisms, and function in sheep horn growth

BackgroundSheep horns play a critical role in the survival and reproduction of sheep. Research on sheep horns not only aids in comprehending their biological roles but is also vital for developing hornless breeds. Although previous studies have suggested that KLK7 may be associated with keratin growth, there are few studies that have focused on the role of KLK7 in sheep horns. This study aims to elucidate the relationship between KLK7 and sheep horns by analyzing the expression, genetic polymorphisms, and potential functions of KLK7 in sheep horns.ResultsThis study utilized RNA sequencing (RNA-Seq) data to analyze the expression levels of the KLK7 gene across different species, as well as among different breeds, tissue types, and genders in sheep. Potential functional sites of KLK7 were explored using whole-genome sequencing (WGS) data. Allele specific expression sites in the KLK7 gene sequence were identified. Finally, the WGS data were linked with sheep horn length for association analysis, and significantly different single-nucleotide polymorphisms (SNPs) were detected and validated by Kompetitive Allele Specific PCR (KASP) genotyping.ConclusionOur results demonstrate that KLK7 was highly expressed in soft horn and skin tissues, and its expression was significantly higher in small-horned sheep than it was in large-horned sheep, suggesting that KLK7 may have an inhibitory effect on horn growth. By comparing the amino acid sequence of KLK7 with KLK7 sequences in other species, we discovered eight amino acids at specific positions in the KLK7 protein sequence that may have regulatory functions in determining the size of horns in ruminant animals. Thirteen SNPs with F-statistic value (Fst) > 0.15 were identified. By integrating RNA-Seq and WGS data, we discovered two SNPs (g.56695395 T > C and g.56695484A > C) with allele specific expression between the large- and small-horned sheep. The two SNPs were validated and were found to have significantly different (P < 0.05) effects on horn length. Our findings suggest a strong association between KLK7 and sheep horn length, indicating the potential role of KLK7 in inhibiting horn growth and providing novel insights into the functionality of KLK7.

Polyploidization-driven transcriptomic dynamics in Medicago sativa neotetraploids: mRNA, smRNA and allele-specific gene expression

Whole genome duplication (WGD) is a powerful evolutionary mechanism in plants. Autopolyploids have been comparatively less studied than allopolyploids, with sexual autopolyploidization receiving even less attention. In this work, we studied the transcriptomes of neotetraploids (2n = 4x = 32) obtained by crossing two diploid (2n = 2x = 16) plants of Medicago sativa that produce a significant percentage of either 2n eggs or pollen. Diploid progeny from the same cross allowed us to separate the transcriptional outcomes of hybridization from those of WGD. This material can help to elucidate events at the base of the domestication of cultivated 4x alfalfa, the world’s most important leguminous forage. Three 2x and three 4x progeny plants and 2x parental plants were used for this study. The RNA-seq data revealed that WGD did not dramatically affect the transcription of leaf protein-coding genes. The two parental genotypes did not contribute equally to the progeny transcriptomes, and genome-wide expression level dominance of the male parent was observed. A large majority of the genes whose expression level changed due to WGD presented increased expression, indicating that the 4x state requires the upregulation of approximately 2.66% of the protein-coding genes. Overall, we estimated that 3.63% of the protein-coding genes were transcriptionally affected by WGD and may contribute to the phenotypic novelty of the neotetraploid plants. Pathway analysis suggested that WGD could affect secondary metabolite biosynthesis, which in turn may influence forage quality. We found four times as many transcription factor genes among the polyploidization-affected genes than among those affected only by hybridization. Several of these belong to classes involved in stress response. Small RNA-seq revealed that very few miRNAs were significantly associated with WGD, but they target several hundred genes, and their role in the WGD response may be relevant. Integrated network analysis led to the identification of putative miRNA: mRNA interactions potentially involved in transcriptome reprogramming. Allele-specific expression analysis indicated that parent-of-origin bias was not a significant outcome of WGD, but we found that parentally biased RNA editing may be a significant source of variation in neopolyploids.

Chromosome-scale and haplotype-resolved genome assembly of Populus trichocarpa

Abstract Populus trichocarpa, a pivotal model organism for woody transgenic research, not only garners substantial scientific interest but plays an integral role in forestry economics. Previous genomic assemblies of P. trichocarpa predominantly treated its heterozygous genome as homozygous, thereby neglecting crucial haplotypic diversity. Leveraging the high fidelity (HiFi) sequencing capabilities of PacBio sequencing and the chromosome conformation capture insights provided by Illumina's Hi-C technique, this study is the first to achieve a near telomere-to-telomere assembly of both paternal and maternal haplotypes in P. trichocarpa. Comparative genomic analysis between these haplotypes has uncovered several allelic variants and pathways critical for trait determination through allele-specific expression. Furthermore, utilizing RNA-seq data from multiple tissues, this investigation has detailed the tissue-specific expression patterns of the leucine-rich repeat (LRR) gene family, which are essential in mediating plant signal transduction and developmental regulation. Our results not only illuminate the functional genomics landscape of P. trichocarpa but also provide invaluable theoretical underpinnings for the genetic improvement of woody plants and a robust framework for exploring genetic variability and allelic expression disparities in arboreal species.

Mechanism of formation of hybrid abalone(Haliotis discus hannai and H. fulgens) heterosis on growth trait based on allele-specific expression analysis

To elucidate the formation mechanism of heterosis in hybrid abalone on growth traits, RNA sequencing was utilized to analyze the transcriptome of hybrid abalone(Haliotis discus hannai ♀ × H. fulgens ♂) with obvious growth differences and some allele-specific expression (ASE) genes related to growth phenotype were identified. The results revealed the following: Nine ASE genes with specific expression were identified in the rapid-growth and slow-growth groups, with six ASE genes unique to the rapid-growth group (CDK13, CROCC, Mfsd2a, Mrps30, MRPL43, NRXN1) and six unique to the slow-growth group (Irgc, OAT, ACVR1, MYLK, MCM3AP, PCDHA8), while three ASE genes were common to both groups (Ttn, DHX8, LOC112566895). The ratio of ASE_SNP reads to total reads in the rapid-growth group tended to be less than 0.5. In contrast, it tended to be greater than 0.5 in the slow-growth group, and individuals in the rapid-growth group exhibited favorable traits toward alternative alleles. In contrast, those in the slow-growth group favored reference alleles. Results suggested that hybrid abalone's superior growth traits tend to originate from the paternal parent (H. fulgens). This study provides insights into gene identification and selection for superior growth traits in hybrid abalone, as well as a theoretical basis for understanding the molecular mechanisms regulating growth traits, thereby advancing the development of abalone genetic improvement and the identification of superior germplasm resources.

A spectrum of nonsense-mediated mRNA decay efficiency along the degree of mutational constraint

Despite its importance for regulating gene expression, nonsense-mediated mRNA decay (NMD) remains poorly understood. Here, we extend the findings of a previous landmark study that proposed several factors associated with NMD efficiency using matched genome and transcriptome data from The Cancer Genome Atlas Program (TCGA) by incorporating additional data including Genotype-Tissue Expression (GTEx), gnomAD, and metrics for mutational constraints. Factors affecting NMD efficiency are analyzed using an allele-specific expression (ASE)-based measure to reduce noise caused by random variations. Additionally, by combining our data with the updated allele frequency database of gnomAD, we demonstrate the spectrum of NMD efficiency according to the degree of gene-level mutational constraints (degree of a gene-tolerating loss-of-function variants). The NMD prediction model, trained on TCGA data, shows that gene-level mutational constraint is an important predictor of NMD efficiency. Findings of this study suggest the potential role of NMD on shaping the landscape of mutational constraints.

Phased T2T genome assemblies facilitate the mining of disease-resistance genes in Vitis davidii

Abstract Grape is an important fruit crop, and its production faces significant threat from diseases, resulting in substantial economic loss. Wild grape relatives are valuable resources for the restoration of disease-resistance loci. However, available resistance loci in wild grape genomes remain largely unexplored. In this study, we assembled two phased genomes, including a high resistant Chinese wild grape, Vitis davidii Föex, and a susceptible cultivar, V. vinifera L. cv. “Manicure Finger”. We detected a total of 36,688 structural variations (SVs), with the genes associated with heterozygous SVs showing an enrichment in allele-specific expression (ASE). Furthermore, we identified eight subgroups of R genes and found that 74.2% of R genes overlap with transposable elements (TEs). Among R genes, NBS-type genes exhibit higher expression profiles in the wild grape genome compared with those in the grape cultivar. Additionally, five specific NBS-type R gene clusters were identified in the wild grape genome that are absent in the cultivar. Through genetic mapping, we identified four quantitative trait loci (QTLs) associated with grape white rot resistance based on the Vitis davidii genome, within which six NBS-type R genes exhibit differential expression between wild and cultivated grapes. Overall, our study revealed the landscape of resistance genes in grape genomes, providing valuable genetic resources for further breeding programs.

Cis-regulatory Variation in Relation to Sex and Sexual Dimorphism in Drosophila melanogaster.

Much of sexual dimorphism is likely due to sex-biased gene expression, which results from differential regulation of a genome that is largely shared between males and females. Here, we use allele-specific expression to explore cis-regulatory variation in Drosophila melanogaster in relation to sex. We develop a Bayesian framework to infer the transcriptome-wide joint distribution of cis-regulatory effects across the sexes. We also examine patterns of cis-regulatory variation with respect to two other levels of variation in sexual dimorphism: (i) across genes that vary in their degree of sex-biased expression and (ii) among tissues that vary in their degree of dimorphism (e.g. relatively low dimorphism in heads vs. high dimorphism in gonads). We uncover evidence of widespread cis-regulatory variation in all tissues examined, with female-biased genes being especially enriched for this variation. A sizeable proportion of cis-regulatory variation is inferred to have sex-specific effects, with sex-dependent cis effects being much more frequent in gonads than in heads. Finally, we find some genes where 1 allele contributes to more than 50% of a gene's expression in heterozygous males but <50% of its expression in heterozygous females. Such variants could provide a mechanism for sex-specific dominance reversals, a phenomenon important for sexually antagonistic balancing selection. However, tissue differences in allelic imbalance are approximately as frequent as sex differences, perhaps suggesting that sexual conflict may not be particularly unique in shaping patterns of expression variation.

Long-read RNA sequencing reveals allele-specific N6-methyladenosine modifications.

Long-read sequencing technology enables highly accurate detection of allele-specific RNA expression, providing insights into the effects of genetic variation on splicing and RNA abundance. Furthermore, the ability to directly sequence RNA using the Oxford Nanopore technology promises the detection of RNA modifications in tandem with ascertaining the allelic origin of each molecule. Here, we leverage these advantages to determine allele-biased patterns of N6-methyladenosine (m6A) modifications in native mRNA. We utilized human and mouse cells with known genetic variants to assign allelic origin of each mRNA molecule combined with a supervised machine learning model to detect read-level m6A modification ratios. Our analyses revealed the importance of sequences adjacent to the DRACH-motif in determining m6A deposition, in addition to allelic differences that directly alter the motif. Moreover, we discovered allele-specific m6A modification (ASM) events with no genetic variants in close proximity to the differentially modified nucleotide, demonstrating the unique advantage of using long reads and surpassing the capabilities of antibody-based short-read approaches. This technological advancement promises to advance our understanding of the role of genetics in determining mRNA modifications.

Genotyping sequence-resolved copy-number variations using pangenomes reveals paralog-specific global diversity and expression divergence of duplicated genes.

Copy-number variable (CNV) genes are important in evolution and disease, yet sequence variation in CNV genes is a blindspot for large-scale studies. We present a method, ctyper, that leverages pangenomes to produce copy-number maps with allele-specific sequences containing locally phased variants of CNV genes from NGS reads. We extensively characterized accuracy and efficiency on a database of 3,351 CNV genes including HLA , SMN , and CYP2D6 as well as 212 non-CNV medically-relevant challenging genes. The genotypes capture 96.5% of underlying variants in new genomes, requiring 0.9 seconds per gene. Expression analysis of ctyper genotypes explains more variance than known eQTL variants. Comparing allele-specific expression quantified divergent expression on 7.94% of paralogs and tissue-specific biases on 4.7% of paralogs. We found reduced expression of SMN-1 converted from SMN-2, which potentially affects diagnosis of spinal muscular atrophy, and increased expression of a duplicative translocation of AMY2B . Overall, ctyper enables biobank-scale genotyping of CNV and challenging genes.

Fusion of breast cancer MCF-7 cells with mesenchymal stem cells rearranges interallelic gene expression and enhances cancer malignancy

Fusion among normal cells is tightly regulated and required for the developmental processes of an organism. Cancer cell fusion appears relatively rare but is associated with generating new hybrid cancer cells with aggressive properties. However, it remains unclear how cancer cells acquire aggressiveness via cell fusion. Here, we report changes in cell proliferative capacity, cell motility, anticancer drug resistance, and gene expression profiles when fusing human MCF-7 breast cancer cells and mesenchymal stem cells (MSCs). The fused cells were established using envelopes of a hemagglutinating virus of Japan, which increased cell proliferation, motility, and drug resistance. Comprehensive gene expression profile analysis revealed that the fused cells expressed higher levels of glycolysis-related genes than their parental cells. In fact, the fused cells relied more on glycolysis for ATP production (Warburg effect). HIF1A, which induces the expression of glycolysis-related genes, was upregulated in fused cells compared to MCF-7 cells. Allele-specific expression analysis of the fused cells indicated that MSC allele-derived HIF1A efficiently induces the expression of glycolysis-related genes in the MCF-7 allele. These findings indicate that the reorganization of gene expression by combining MSCs and MCF-7 alleles resulted in the predominant expression of glycolysis-related genes and increased malignancy in the fused cells.

Candida albicans isolates contain frequent heterozygous structural variants and transposable elements within genes and centromeres.

The human fungal pathogen Candida albicans poses a significant burden on global health, causing high rates of mortality and antifungal drug resistance. C. albicans is a heterozygous diploid organism that reproduces asexually. Structural variants (SVs) are an important source of genomic rearrangement, particularly in species that lack sexual recombination. To comprehensively investigate SVs across clinical isolates of C. albicans, we conducted long read sequencing and genome-wide SV analysis in three distantly related clinical isolates. Our work included a new, comprehensive analysis of transposable element (TE) composition, location and diversity. SVs and TEs are frequently close to coding sequences and many SVs are heterozygous, suggesting that SVs might impact gene and allele-specific expression. Most SVs are uniquely present in only one clinical isolate, indicating that SVs represent a significant source of intra-species genetic variation. We identified multiple, distinct SVs at the centromeres of Chromosome 4 and Chromosome 5, including inversions and transposon polymorphisms. These two chromosomes are often aneuploid in drug resistant clinical isolates, and can form isochromosome structures with breakpoints near the centromere. Further screening of 100 clinical isolates confirmed the widespread presence of centromeric SVs in C. albicans, often appearing in a heterozygous state, indicating that SVs are contributing to centromere evolution in C. albicans Together, these findings highlight that SVs and TEs are common across diverse clinical isolates of C. albicans and that the centromeres of this organism are important sites of genome rearrangement.

Stabilizing selection and adaptation shape cis and trans gene expression variation in C. elegans.

An outstanding question in the evolution of gene expression is the relative influence of neutral processes versus natural selection, including adaptive change driven by directional selection as well as stabilizing selection, which may include compensatory dynamics. These forces shape patterns of gene expression variation within and between species, including the regulatory mechanisms governing expression in cis and trans. In this study, we interrogate intraspecific gene expression variation among seven wild C. elegans strains, with varying degrees of genomic divergence from the reference strain N2, leveraging this system's unique advantages to comprehensively evaluate gene expression evolution. By capturing allele-specific and between-strain changes in expression, we characterize the regulatory architecture and inheritance mode of gene expression variation within C. elegans and assess their relationship to nucleotide diversity, genome evolutionary history, gene essentiality, and other biological factors. We conclude that stabilizing selection is a dominant influence in maintaining expression phenotypes within the species, and the discovery that genes with higher overall expression tend to exhibit fewer expression differences supports this conclusion, as do widespread instances of cis differences compensated in trans. Moreover, analyses of human expression data replicate our finding that higher expression genes have less variable expression. We also observe evidence for directional selection driving expression divergence, and that expression divergence accelerates with increasing genomic divergence. To provide community access to the data from this first analysis of allele-specific expression in C. elegans, we introduce an interactive web application, where users can submit gene-specific queries to view expression, regulatory pattern, inheritance mode, and other information: https://wildworm.biosci.gatech.edu/ase/.

Trans regulation of an odorant binding protein by a proto-Y chromosome affects male courtship in house fly.

The male-limited inheritance of Y chromosomes favors alleles that increase male fitness, often at the expense of female fitness. Determining the mechanisms underlying these sexually antagonistic effects is challenging because it can require studying Y-linked alleles while they still segregate as polymorphisms. We used a Y chromosome polymorphism in the house fly, Musca domestica, to address this challenge. Two male determining Y chromosomes (YM and IIIM) segregate as stable polymorphisms in natural populations, and they differentially affect multiple traits, including male courtship performance. We identified differentially expressed genes encoding odorant binding proteins (in the Obp56h family) as candidate agents for the courtship differences. Through network analysis and allele-specific expression measurements, we identified multiple genes on the house fly IIIM chromosome that could serve as trans regulators of Obp56h gene expression. One of those genes is homologous to Drosophila melanogaster CG2120, which encodes a transcription factor that binds near Obp56h. Upregulation of CG2120 in D. melanogaster nervous tissues reduces copulation latency, consistent with this transcription factor acting as a negative regulator of Obp56h expression. The transcription factor gene, which we name speed date, demonstrates a molecular mechanism by which a Y-linked gene can evolve male-beneficial effects.

Long-read sequencing of an advanced cancer cohort resolves rearrangements, unravels haplotypes, and reveals methylation landscapes

The Long-Read Personalized OncoGenomics (POG) dataset comprises a cohort of 189 patient tumors and 41 matched normal samples sequenced using the Oxford Nanopore Technologies PromethION platform. This dataset from the POG program and the Marathon of Hope Cancer Centres Network includes DNA and RNA short-read sequence data, analytics, and clinical information. We show the potential of long-read sequencing for resolving complex cancer-related structural variants, viral integrations, and extrachromosomal circular DNA. Long-range phasing facilitates the discovery of allelically differentially methylated regions (aDMRs) and allele-specific expression, including recurrent aDMRs in the cancer genes RET and CDKN2A. Germline promoter methylation in MLH1 can be directly observed in Lynch syndrome. Promoter methylation in BRCA1 and RAD51C is a likely driver behind homologous recombination deficiency where no coding driver mutation was found. This dataset demonstrates applications for long-read sequencing in precision medicine and is available as a resource for developing analytical approaches using this technology.

Linking candidate causal autoimmune variants to T cell networks using genetic and epigenetic screens in primary human T cells.

Genetic variants associated with autoimmune diseases are highly enriched within putative cis -regulatory regions of CD4 + T cells, suggesting that they alter disease risk via changes in gene regulation. However, very few genetic variants have been shown to affect T cell gene expression or function. We tested >18,000 autoimmune disease-associated variants for allele-specific expression using massively parallel reporter assays in primary human CD4 + T cells. The 545 expression-modulating variants (emVars) identified greatly enrich for likely causal variants. We provide evidence that many emVars are mediated by common upstream regulatory conduits, and that putative target genes of primary T cell emVars are highly enriched within a lymphocyte activation network. Using bulk and single-cell CRISPR-interference screens, we confirm that emVar-containing T cell cis -regulatory elements modulate both known and novel target genes that regulate T cell proliferation, providing plausible mechanisms by which these variants alter autoimmune disease risk.

Characterizing the allele-specific gene expression landscape in high hyperdiploid acute lymphoblastic leukemia with BASE.

Somatic copy number variations (CNVs), including abnormal chromosome numbers and structural changes leading to gain or loss of genetic material, play a crucial role in initiation and progression of cancer. CNVs are believed to cause gene dosage imbalances and modify cis-regulatory elements, leading to allelic expression imbalances in genes that influence cell division and thereby contribute to cancer development. However, the impact of CNVs on allelic gene expression in cancer remains unclear. Allele-specific expression (ASE) analysis, a potent method for investigating genome-wide allelic imbalance profiles in tumors, assesses the relative expression of two alleles using high-throughput sequencing data. However, many existing methods for gene-level ASE detection rely on only RNA sequencing data, which present challenges in interpreting the genetic mechanisms underlying ASE in cancer. To address this issue, we developed a robust framework that integrates allele-specific copy number calls into ASE calling algorithms by leveraging paired genome and transcriptome data from the same sample. This integration enhances the interpretability of the genetic mechanisms driving ASE, thereby facilitating the identification of driver events triggered by CNVs in cancer. In this study, we utilized BASE to conduct a comprehensive analysis of ASE in high hyperdiploid acute lymphoblastic leukemia (HeH ALL), a prevalent childhood malignancy characterized by gains of chromosomes X, 4, 6, 10, 14, 17, 18, and 21. Our analysis unveiled the comprehensive ASE landscape in HeH ALL. Through a multi-perspective examination of HeH ASEs, we offer a systematic understanding of how CNVs impact ASE in HeH, providing valuable insights to guide ASE studies in cancer.

Promoter Deletion Leading to Allele Specific Expression in a Genetically Unsolved Case of Primary Ciliary Dyskinesia.

Variation in the non-coding genome represents an understudied mechanism of disease and it remains challenging to predict if single nucleotide variants, small insertions and deletions, or structural variants in non-coding genomic regions will be detrimental. Our approach using complementary RNA-seq and targeted long-read DNA sequencing can prioritize identification of non-coding variants that lead to disease via alteration of gene splicing or expression. We have identified a patient with primary ciliary dyskinesia with a pathogenic coding variant on one allele of the SPAG1 gene, while the second allele appears normal by whole exome sequencing despite an autosomal recessive inheritance pattern. RNA sequencing revealed reduced SPAG1 transcript levels and exclusive allele specific expression of the known pathogenic allele, suggesting the presence of a non-coding variant on the second allele that impacts transcription. Targeted long-read DNA sequencing identified a heterozygous 3 kilobase deletion of the 5' untranslated region of SPAG1, overlapping the promoter and first non-coding exon. This non-coding deletion was missed by whole exome sequencing and gene-specific deletion/duplication analysis, highlighting the importance of investigating the non-coding genome in patients with "missing" disease-causing variation. This paradigm demonstrates the utility of both RNA and long-read DNA sequencing in identifying pathogenic non-coding variants in patients with unexplained genetic disease.

Maintenance of X chromosome inactivation after T cell activation requires NF-κB signaling.

X chromosome inactivation (XCI) balances X-linked gene dosage between sexes. Unstimulated T cells lack cytological enrichment of X-inactive specific transcript (Xist) RNA and heterochromatic modifications on the inactive X chromosome (Xi), which are involved in maintenance of XCI, and these modifications return to the Xi after stimulation. Here, we examined allele-specific gene expression and epigenomic profiles of the Xi in T cells. We found that the Xi in unstimulated T cells is largely dosage compensated and enriched with the repressive H3K27me3 modification but not the H2AK119-ubiquitin (Ub) mark. Upon T cell stimulation mediated by both CD3 and CD28, the Xi accumulated H2AK119-Ub at gene regions of previous H3K27me3 enrichment. T cell receptor (TCR) engagement, specifically NF-κB signaling downstream of the TCR, was required for Xist RNA localization to the Xi. Disruption of NF-κB signaling in mouse and human T cells using genetic deletion, chemical inhibitors, and patients with immunodeficiencies prevented Xist/XIST RNA accumulation at the Xi and altered X-linked gene expression. Our findings reveal a previously undescribed connection between NF-κB signaling pathways, which affects XCI maintenance in T cells in females.