Allelic Expression Imbalance Research Articles

Integrative Analysis Identified rs907091 at the miRNA Binding Region of the IKZF3 Gene as a Potential Functional Variant for Rheumatoid Arthritis

Objective: Previous studies have identified a large number of associations between genetic variants and rheumatoid arthritis (RA), but the functional mechanisms underlying the associations are largely unknown. Methods: Based on publicly available datasets and results, we explored the functional mechanisms underlying the associations between regulatory polymorphisms in miRNA target sites (poly-miRTSs) and RA by combining integrative analyses (expression quantitative trait locus analysis, eQTL, and gene enrichment analysis) with in-house molecular experiments (allelic expression imbalance experiment, AEI, and dual-luciferase reporter gene assay). Results: By integrating the results from the MirSNP and eQTL databases, a total of 114 pairs between poly-miRTSs and target genes were identified, which correspond to 70 unique poly-miRTSs and 28 genes. Most of the 28 genes were located in the HLA region, and they tended to be enriched in multiple immune-related GO terms or pathways, e.g., “antigen processing and presentation” and “immune response”. Among these poly-miRTSs, rs907091 in the 3’UTR of IKZF3 (non-HLA region) was highlighted. Further AEI experiments showed that the C allele had a higher expression of ~18% than the T allele, suggesting an obvious allelic expression imbalance. Dual-luciferase reporter gene assays showed that miR-326 and miR-330-5p could regulate IKZF3 expression (P<0.05) mediated by rs907091 in the 3’UTR binding site. Conclusion: Our study explored some functional mechanisms underlying the associations between poly-miRTSs and target genes and highlighted rs907091 at the miRNA binding region of the IKZF3 gene as a potential functional variant for rheumatoid arthritis.

Inter-subspecies mouse F1 hybrid embryonic stem cell lines newly established for studies of allelic imbalance in gene expression.

Allele-specific monoallelic gene expression is a unique phenomenon and a great resource for analyzing gene regulation. To study this phenomenon, we established new embryonic stem (ES) cell lines derived from F1 hybrid blastocysts from crosses between four mouse subspecies (Mus musculus domesticus, C57BL/6; M. musculus molossinus, MSM/Ms; M. musculus musculus, PWK; M. musculus castaneus, HMI/Ms) and analyzed the expression levels of undifferentiated pluripotent stem cell markers and karyotypes of each line. To demonstrate the utility of our cell lines, we analyzed the allele-specific expression pattern of the Inpp5d gene as an example. The allelic expression depended on the parental alleles; this dependence could be a consequence of differences in compatibility between cis- and trans-elements of the Inpp5d gene from different subspecies. The use of parental mice from four subspecies greatly enhanced genetic polymorphism. The F1 hybrid ES cells retained this polymorphism not only in the Inpp5d gene, but also at a genome-wide level. As we demonstrated for the Inpp5d gene, the established cell lines can contribute to the analysis of allelic expression imbalance based on the incompatibility between cis- and trans-elements and of phenotypes related to this incompatibility.

Heterozygous CARD9 mutation favors the development of allergic bronchopulmonary aspergillosis.

Previous research demonstrated that a homozygous mutation of g.136372044G>A (S12N) in caspase recruitment domain family member 9 ( CARD9 ) is critical for producing Aspergillus fumigatus -induced ( Af -induced) T helper 2 (T H 2)-mediated responses in allergic bronchopulmonary aspergillosis (ABPA). However, it remains unclear whether the CARD9S12N mutation, especially the heterozygous occurrence, predisposes the host to ABPA. A total of 61 ABPA patients and 264 controls (including 156 healthy controls and 108 asthma patients) were recruited for sequencing the CARD9 locus to clarify whether patients with this heterozygous single-nucleotide polymorphisms are predisposed to the development of ABPA. A series of in vivo and in vitro experiments, such as quantitative real-time polymerase chain reaction, flow cytometry, and RNA isolation and quantification, were used to illuminate the involved mechanism of the disease. The presence of the p.S12N mutation was associated with a significant risk of ABPA in ABPA patients when compared with healthy controls and asthma patients, regardless of Aspergillus sensitivity. Relative to healthy controls without relevant allergies, the mutation of p.S12N was associated with a significant risk of ABPA (OR: 2.69 and 4.17 for GA and AA genotypes, P = 0.003 and 0.029, respectively). Compared with patients with asthma, ABPA patients had a significantly higher heterozygous mutation (GA genotype), indicating that p.S12N might be a significant ABPA-susceptibility locus ( aspergillus sensitized asthma: OR: 3.02, P = 0.009; aspergillus unsensitized asthma: OR: 2.94, P = 0.005). The mutant allele was preferentially expressed in ABPA patients with heterozygous CARD9S12N , which contributes to its functional alterations to facilitate Af -induced T H 2-mediated ABPA development. In terms of mechanism, Card9 wild-type ( Card9WT ) expression levels decreased significantly due to Af -induced decay of its messenger RNA compared to the heterozygous Card9S12N . In addition, ABPA patients with heterozygous CARD9S12N had increased Af -induced interleukin-5 production. Our study provides the genetic evidence showing that the heterozygous mutation of CARD9S12N , followed by allele expression imbalance of CARD9S12N , facilitates the development of ABPA.

The phased chromosome-scale genome of yellowhorn sheds light on the mechanism of petal color change

Yellowhorn (Xanthoceras sorbifolium), especially its varieties, is the red petal yellowhorn (X. sorbifolium var. purpurea), an important tree species with great ornamental value, and its flower petals change color throughout the flowering period. In this study, we mainly focused on the mechanism of the petal color change with transcriptomics and metabolomics data. A phased chromosome-scale assembly of the red petal yellowhorn genome was generated using the PacBio high-fidelity reads, Illumina short reads, and Phase genomics Proximo Hi-C data. The final de novo assembly yielded 533.67 Mb with a contig N50 of 5.42 Mb, and 27 501 protein-coding genes were predicted. Notably, an alternate haplotig assembly was also obtained. Furthermore, a variation database for the alleles within the genome was constructed. Subsequently, the expression pattern of flower pigmentation-related genes and allelic expression imbalance events were investigated. Apart from 6 genes involved in the anthocyanin biosynthesis pathway regulated by the activation of 15 MYB-bHLH-WD40s, the increased expression of senescence-related gene 1 (SRG1) and 2-oxoglutarate-dependent dioxygenase (DIOX5) might also result in decreasing content of lutein and increasing abundance of (E/Z)-phytoene, cyanidin-3-O-rutinoside, and cyanidin-3-O-sambubioside. These changes in genes and metabolites were most likely related to the petal color change in red petal yellowhorn. This phased chromosome-scale genome assembly provides more accurate genomic information for future molecular breeding and facilitates allele function studies of the red petal yellowhorn.

Mediation of the Same Epigenetic and Transcriptional Effect by Independent Osteoarthritis Risk-Conferring Alleles on a Shared Target Gene, COLGALT2.

Over 100 DNA variants have been associated with osteoarthritis (OA), including rs1046934, located within a linkage disequilibrium block encompassing part of COLGALT2 and TSEN15. The present study was undertaken to determine the target gene(s) and the mechanism of action of the OA locus using human fetal cartilage, cartilage from OA and femoral neck fracture arthroplasty patients, and a chondrocyte cell model. Genotyping and methylation array data of DNA from human OA cartilage samples (n=87) were used to determine whether the rs1046934 genotype is associated with differential DNA methylation at proximal CpGs. Results were replicated in DNA from human arthroplasty (n=132) and fetal (n=77) cartilage samples using pyrosequencing. Allelic expression imbalance (AEI) measured the effects of genotype on COLGALT2 and TSEN15 expression. Reporter gene assays and epigenetic editing determined the functional role of regions harboring differentially methylated CpGs. In silico analyses complemented these experiments. Three differentially methylated CpGs residing within regulatory regions were detected in the human OA cartilage array data, and 2 of these were replicated in human arthroplasty and fetal cartilage. AEI was detected for COLGALT2 and TSEN15, with associations between expression and methylation for COLGALT2. Reporter gene assays confirmed that the CpGs are in chondrocyte enhancers, with epigenetic editing results directly linking methylation with COLGALT2 expression. COLGALT2 is a target of this OA locus. We previously characterized another OA locus, marked by rs11583641, that independently targets COLGALT2. The genotype of rs1046934, like rs11583641, mediates its effect by modulating expression of COLGALT2 via methylation changes to CpGs located in enhancers. Although the single-nucleotide polymorphisms, CpGs, and enhancers are distinct between the 2 independent OA risk loci, their effect on COLGALT2 is the same. COLGALT2 is the target of independent OA risk loci sharing a common mechanism of action.

Expression of INPP5D Isoforms in Human Brain: Impact of Alzheimer's Disease Neuropathology and Genetics.

The single nucleotide polymorphisms rs35349669 and rs10933431 within Inositol Polyphosphate-5-Phosphatase D (INPP5D) are strongly associated with Alzheimer's Disease risk. To better understand INPP5D expression in the brain, we investigated INPP5D isoform expression as a function of rs35349669 and rs10933431, as well as Alzheimer's disease neuropathology, by qPCR and isoform-specific primers. In addition, INPP5D allelic expression imbalance was evaluated relative to rs1141328 within exon 1. Expression of INPP5D isoforms associated with transcription start sites in exon 1 and intron 14 was increased in individuals with high Alzheimer's disease neuropathology. In addition, a novel variant with 47bp lacking from exon 12 increased expression in Alzheimer's Disease brains, accounting for 13% of total INPP5D expression, and was found to undergo nonsense-mediated decay. Although inter-individual variation obscured a possible polymorphism effect on INPP5D isoform expression as measured by qPCR, rs35349669 was associated with rs1141328 allelic expression imbalance, suggesting that rs35349669 is significantly associated with full-length INPP5D isoform expression. In summary, expression of INPP5D isoforms with start sites in exon 1 and intron 14 are increased in brains with high Alzheimer's Disease neuropathology, a novel isoform lacking the phosphatase domain was significantly increased with the disease, and the polymorphism rs35349669 correlates with allele-specific full-length INPP5D expression.

Genetic analysis and allele-specific expression of SMAD7 3′UTR variants in human colorectal cancer reveal a novel somatic variant exhibiting allelic imbalance

BackgroundConsidering the impact of SMAD7 deregulation in colorectal cancer (CRC) progression and the significance of single nucleotide variant (SNV)-mediated disruptions of microRNA (miRNA)-dependent regulation for cancer susceptibility, our study aimed to analyze genetic variation in the SMAD7 3′ untranslated region ( 3′UTR) in CRC, measure differences in allelic mRNA expression, and evaluate its interference with miRNA-mediated post-transcriptional regulation. Patients and MethodsThis study included 80 patients with different CRC stages and six human colon cancer cell lines of various histological origins. SMAD7 3′UTR was analyzed by direct sequencing, followed by the relative quantification of differential allelic expression of detected variants by allele-specific qRT-PCR. In silico tools were employed for predictions of regulatory consequences of detected variants. ResultsA total of four different SNVs in one cell line and nine patients were found, among which were a novel somatic point variant and three already known germline variants (rs16950113, rs1050799536, and rs1043778717). All evaluated SNVs exhibited variable extents of allelic imbalance in expression. In silico analysis predicted significant effects of SNVs on miRNA binding efficiency, with each SNV disrupting existing and creating new target sites for one or more miRNAs. ConclusionImbalance observed in the expression of SNV alleles altering miRNA binding suggests that all investigated SNVs are potential contributing factors impacting SMAD7 expression regulation in CRC that further studies should investigate.

Diagnostic and therapeutic potential of RNASET2 in Crohn's disease: Disease-risk polymorphism modulates allelic-imbalance in expression and circulating protein levels and recombinant-RNASET2 attenuates pro-inflammatory cytokine secretion.

Ribonuclease T2 gene (RNASET2) variants are associated in genome wide association studies (GWAS) with risk for several autoimmune diseases, including Crohn's disease (CD). In T cells, a functional and biological relationship exists between TNFSF15-mediated enhancement of IFN-γ production, mucosal inflammation and RNASET2. Disease risk variants are associated with decreased mRNA expression and clinical characteristics of severe CD; however, functional classifications of variants and underlying molecular mechanisms contributing to pathogenesis remain largely unknown. In this study we demonstrate that allelic imbalance of RNASET2 disease risk variant rs2149092 is associated with transcriptional and post-transcriptional mechanisms regulating transcription factor binding, promoter-transactivation and allele-specific expression. RNASET2 mRNA expression decreases in response to multiple modes of T cell activation and recovers following elimination of activator. In CD patients with severe disease necessitating surgical intervention, preoperative circulating RNASET2 protein levels were decreased compared to non-IBD subjects and rebounded post-operatively following removal of the inflamed region, with levels associated with allelic carriage. Furthermore, overexpression or treatment with recombinant RNASET2 significantly reduced IFN-γ secretion. These findings reveal that RNASET2 cis- and trans-acting variation contributed regulatory complexity and determined expression and provide a basis for linking genetic variation with CD pathobiology. These data may ultimately identify RNASET2 as an effective therapeutic target in a subset of CD patients with severe disease.

Allelic expression imbalance in articular cartilage and subchondral bone refined genome-wide association signals in osteoarthritis.

To present an unbiased approach to identify positional transcript single nucleotide polymorphisms (SNPs) of osteoarthritis (OA) risk loci by allelic expression imbalance (AEI) analyses using RNA sequencing of articular cartilage and subchondral bone from OA patients. RNA sequencing from 65 articular cartilage and 24 subchondral bone from OA patients was used for AEI analysis. AEI was determined for all genes present in the 100 regions reported by the genome-wide association studies (GWAS) catalog that were also expressed in cartilage or bone. The count fraction of the alternative allele (φ) was calculated for each heterozygous individual with the risk SNP or with the SNP in linkage disequilibrium (LD) with it (r2 > 0.6). Furthermore, a meta-analysis was performed to generate a meta-φ (null hypothesis median φ = 0.49) and P-value for each SNP. We identified 30 transcript SNPs (28 in cartilage and two in subchondral bone) subject to AEI in 29 genes. Notably, 10 transcript SNPs were located in genes not previously reported in the GWAS catalog, including two long intergenic non-coding RNAs (lincRNAs), MALAT1 (meta-φ = 0.54, FDR = 1.7×10-4) and ILF3-DT (meta-φ = 0.6, FDR = 1.75×10-5). Moreover, 12 drugs were interacting with seven genes displaying AEI, of which seven drugs have been already approved. By prioritizing proxy transcript SNPs that mark AEI in cartilage and/or subchondral bone at loci harbouring GWAS signals, we present an unbiased approach to identify the most likely functional OA risk-SNP and gene. We identified 10 new potential OA risk genes ready for further translation towards underlying biological mechanisms.

In Vivo Clonal Analysis Reveals Random Monoallelic Expression in Lymphocytes That Traces Back to Hematopoietic Stem Cells.

Evaluating the epigenetic landscape in the stem cell compartment at the single-cell level is essential to assess the cells’ heterogeneity and predict their fate. Here, using a genome-wide transcriptomics approach in vivo, we evaluated the allelic expression imbalance in the progeny of single hematopoietic cells (HSCs) as a read-out of epigenetic marking. After 4 months of extensive proliferation and differentiation, we found that X-chromosome inactivation (XCI) is tightly maintained in all single-HSC derived hematopoietic cells. In contrast, the vast majority of the autosomal genes did not show clonal patterns of random monoallelic expression (RME). However, a persistent allele-specific autosomal transcription in HSCs and their progeny was found in a rare number of cases, none of which has been previously reported. These data show that: 1) XCI and RME in the autosomal chromosomes are driven by different mechanisms; 2) the previously reported high frequency of genes under RME in clones expanded in vitro (up to 15%) is not found in clones undergoing multiple differentiation steps in vivo; 3) prior to differentiation, HSCs have stable patterns of autosomal RME. We propose that most RME patterns in autosomal chromosomes are erased and established de novo during cell lineage differentiation.

Abstract 1213: Uncovering HLA loss of heterozygosity and allelic imbalance in cancer for the improvement of personalized neoTCR immunotherapy with PACT-ESCAPE

Abstract Introduction: PACT Pharma is a clinical-stage adoptive cell therapy company that develops personalized neoTCR-T cells using a state-of-the-art approach to discover and validate predicted neoepitopes and their cognate T cell receptors (clinical trial NCT03970382). An important mechanism of resistance to adoptive cell therapy targeting tumor-specific neoantigens is the genetic loss of human leukocyte antigen (HLA) alleles or alterations in HLA expression in tumor cells. Therapies targeting neoantigens presented by HLA alleles that have either been deleted, mutated or are strongly downregulated are likely to lack efficacy due to the absence or reduction of epitope presentation. Surveying neoepitope presentation escape mechanisms is key for personalized immunotherapy. Methods: PACT Pharma has developed PACT-ESCAPE, a method that integrates DNA and RNA sequencing data to quantify allelic imbalance at HLA loci, including the most extreme case of loss of heterozygosity (LOH). Allelic imbalance is first determined at the DNA level by determining the patient’s maternal and paternal haplotypes across chromosome 6p to infer their copy number states. Twenty-six HLA Class I and II loci from chromosome 6 are then genotyped at high resolution, enabling accurate assignment of copy number states to the patient’s alleles, including any alleles that have been deleted in the tumor. Unlike other currently available methods, PACT-ESCAPE also measures the relative RNA expression of HLA alleles to provide an orthogonal estimate of allelic imbalance across chromosome 6 and support loss of heterozygosity calls made at the DNA level. Presentation machinery genes are also surveyed for loss of function mutations. To benchmark PACT-ESCAPE, we compared loss of heterozygosity calls at HLA-A, HLA-B, and HLA-C between our method and a previously published DNA-based HLA LOH classifier for 17 samples for which we had matched tumor/normal whole exome sequencing. Results: We found 100% concordance between PACT-ESCAPE and the benchmark, with four samples showing LOH at HLA-A, HLA-B, and HLA-C at the DNA level. Next, we evaluated RNA sequencing from tumor samples and confirmed that the relative expression of the lost allele compared to the kept allele was lower when LOH was called. High levels of allelic imbalance in gene expression, however, were not unique to samples with LOH, suggesting that differential expression of HLA alleles might also be an important contributor to immune escape in cancer. Conclusions: Evaluation of allelic imbalance in DNA copy number and RNA expression in PACT-ESCAPE provides new insights into immune escape in cancer. Adoptive cell therapy targeting neoantigens will benefit by enabling the accurate selection of targets most likely to be presented by the tumor. Citation Format: Chad C. Smith, Yan Ma, Katie Campbell, Zheng Pan, Eric Stawiski. Uncovering HLA loss of heterozygosity and allelic imbalance in cancer for the improvement of personalized neoTCR immunotherapy with PACT-ESCAPE [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1213.

Allelic expression imbalance of PIK3CA mutations is frequent in breast cancer and prognostically significant.

PIK3CA mutations are the most common in breast cancer, particularly in the estrogen receptor-positive cohort, but the benefit of PI3K inhibitors has had limited success compared with approaches targeting other less common mutations. We found a frequent allelic expression imbalance between the missense mutant and wild-type PIK3CA alleles in breast tumors from the METABRIC (70.2%) and the TCGA (60.1%) projects. When considering the mechanisms controlling allelic expression, 27.7% and 11.8% of tumors showed imbalance due to regulatory variants in cis, in the two studies respectively. Furthermore, preferential expression of the mutant allele due to cis-regulatory variation is associated with poor prognosis in the METABRIC tumors (P = 0.031). Interestingly, ER−, PR−, and HER2+ tumors showed significant preferential expression of the mutated allele in both datasets. Our work provides compelling evidence to support the clinical utility of PIK3CA allelic expression in breast cancer in identifying patients of poorer prognosis, and those with low expression of the mutated allele, who will unlikely benefit from PI3K inhibitors. Furthermore, our work proposes a model of differential regulation of a critical cancer-promoting gene in breast cancer.

Interpreting coronary artery disease GWAS results: A functional genomics approach assessing biological significance.

Genome-wide association studies (GWAS) have implicated 58 loci in coronary artery disease (CAD). However, the biological basis for these associations, the relevant genes, and causative variants often remain uncertain. Since the vast majority of GWAS loci reside outside coding regions, most exert regulatory functions. Here we explore the complexity of each of these loci, using tissue specific RNA sequencing data from GTEx to identify genes that exhibit altered expression patterns in the context of GWAS-significant loci, expanding the list of candidate genes from the 75 currently annotated by GWAS to 245, with almost half of these transcripts being non-coding. Tissue specific allelic expression imbalance data, also from GTEx, allows us to uncover GWAS variants that mark functional variation in a locus, e.g., rs7528419 residing in the SORT1 locus, in liver specifically, and rs72689147 in the GUYC1A1 locus, across a variety of tissues. We consider the GWAS variant rs1412444 in the LIPA locus in more detail as an example, probing tissue and transcript specific effects of genetic variation in the region. By evaluating linkage disequilibrium (LD) between tissue specific eQTLs, we reveal evidence for multiple functional variants within loci. We identify 3 variants (rs1412444, rs1051338, rs2250781) that when considered together, each improve the ability to account for LIPA gene expression, suggesting multiple interacting factors. These results refine the assignment of 58 GWAS loci to likely causative variants in a handful of cases and for the remainder help to re-prioritize associated genes and RNA isoforms, suggesting that ncRNAs maybe a relevant transcript in almost half of CAD GWAS results. Our findings support a multi-factorial system where a single variant can influence multiple genes and each genes is regulated by multiple variants.

Identification of TMEM129, encoding a ubiquitin-protein ligase, as an effector gene of osteoarthritis genetic risk

BackgroundOsteoarthritis is highly heritable and genome-wide studies have identified single nucleotide polymorphisms (SNPs) associated with the disease. One such locus is marked by SNP rs11732213 (T > C). Genotype at rs11732213 correlates with the methylation levels of nearby CpG dinucleotides (CpGs), forming a methylation quantitative trait locus (mQTL). This study investigated the regulatory activity of the CpGs to identify a target gene of the locus.MethodsNucleic acids were extracted from the articular cartilage of osteoarthritis patients. Samples were genotyped, and DNA methylation was quantified by pyrosequencing at 14 CpGs within a 259-bp interval. CpGs were tested for enhancer effects in immortalised chondrocytes using a reporter gene assay. DNA methylation at the locus was altered using targeted epigenome editing, with the impact on gene expression determined using quantitative polymerase chain reaction.Resultsrs11732213 genotype correlated with DNA methylation at nine CpGs, which formed a differentially methylated region (DMR), with the osteoarthritis risk allele T corresponding to reduced levels of methylation. The DMR acted as an enhancer and demethylation of the CpGs altered expression of TMEM129. Allelic imbalance in TMEM129 expression was identified in cartilage, with under-expression of the risk allele.ConclusionsTMEM129 is a target of osteoarthritis genetic risk at this locus. Genotype at rs11732213 impacts DNA methylation at the enhancer, which, in turn, modulates TMEM129 expression. TMEM129 encodes an enzyme involved in protein degradation within the endoplasmic reticulum, a process previously implicated in osteoarthritis. TMEM129 is a compelling osteoarthritis susceptibility target.

Abstract 10651: Identification of Allele-Specific Gene Expression in Association with Blood Pressure

Introduction: Risk of hypertension is explained by both behavioral and genetic factors that may act on transcriptomic mechanisms to affect blood pressure variability. Methods: Whole genome sequencing and RNA sequencing data were obtained on 1,671 Framingham Heart Study (FHS) participants (mean age 54±13 years). Allelic expression of heterozygous SNPs was measured and allelic imbalance was identified using a binomial test. SNPs with significant allelic imbalance were evaluated in linear regression to test the association between the reference/total allele count ratio and 1) systolic blood pressure (SBP), and 2) diastolic blood pressure (DBP). Models were adjusted for age, sex, BMI, and family structure. Results: At a Bonferroni-corrected p-value<6.4E-8, we identified 20,890 SNPs with significant allelic imbalance at MAF>0.01. Prior GWAS have identified >2,000 SNPs associated with SBP, DBP, or hypertension; we identified allelic expression imbalance of 60 SNPs (in 44 genes) that were reported in GWAS to be associated with SBP, and of eight SNPs (in two genes) that are associated with DBP. These genes were significantly enriched for the immune effector process pathway (p=1.4E-7). Fifteen SNPs were located in promoter or promoter-flanking regions, while the remaining SNPs were located in protein-coding regions. Seventeen SNPs were also cis -eQTLs, of which four transcripts also were associated with SBP or DBP in the FHS cohort. Conclusion: We identified ASE linked to blood pressure. Our results suggested that novel transcriptomic regulatory mechanisms involving allelic imbalance at specific SNPs may play a role in blood pressure regulation.

Locus specific epigenetic modalities of random allelic expression imbalance

Most autosomal genes are thought to be expressed from both alleles, with some notable exceptions, including imprinted genes and genes showing random monoallelic expression (RME). The extent and nature of RME has been the subject of debate. Here we investigate the expression of several candidate RME genes in F1 hybrid mouse cells before and after differentiation, to define how they become persistently, monoallelically expressed. Clonal monoallelic expression is not present in embryonic stem cells, but we observe high frequencies of monoallelism in neuronal progenitor cells by assessing expression status in more than 200 clones. We uncover unforeseen modes of allelic expression that appear to be gene-specific and epigenetically regulated. This non-canonical allelic regulation has important implications for development and disease, including autosomal dominant disorders and opens up therapeutic perspectives.

The CDH1 c.1901C>T Variant: A Founder Variant in the Portuguese Population with Severe Impact in mRNA Splicing.

Simple SummaryAn unexpectedly high number of early-onset diffuse gastric and lobular breast cancer in apparently unrelated families carrying the same CDH1 c.1901C>T variant (formerly known as missense p.A634V) in Northern Portugal suggested a founder effect in this region. We demonstrated that c.1901C>T is a truncating variant triggered by cryptic splicing, calculated its mutational age, and characterized the tumour spectrum and age of onset in affected families.Hereditary diffuse gastric cancer (HDGC) caused by CDH1 variants predisposes to early-onset diffuse gastric (DGC) and lobular breast cancer (LBC). In Northern Portugal, the unusually high number of HDGC cases in unrelated families carrying the c.1901C>T variant (formerly known as p.A634V) suggested this as a CDH1-founder variant. We aimed to demonstrate that c.1901C>T is a bona fide truncating variant inducing cryptic splicing, to calculate the timing of a potential founder effect, and to characterize tumour spectrum and age of onset in carrying families. The impact in splicing was proven by using carriers’ RNA for PCR-cloning sequencing and allelic expression imbalance analysis with SNaPshot. Carriers and noncarriers were haplotyped for 12 polymorphic markers, and the decay of haplotype sharing (DHS) method was used to estimate the time to the most common ancestor of c.1901C>T. Clinical information from 58 carriers was collected and analysed. We validated the cryptic splice site within CDH1-exon 12, which was preferred over the canonical one in 100% of sequenced clones. Cryptic splicing induced an out-of-frame 37bp deletion in exon 12, premature truncation (p.Ala634ProfsTer7), and consequently RNA mediated decay. The haplotypes carrying the c.1901C>T variant were found to share a common ancestral estimated at 490 years (95% Confidence Interval 445–10,900). Among 58 carriers (27 males (M)–31 females (F); 13–83 years), DGC occurred in 11 (18.9%; 4M–7F; average age 33 ± 12) and LBC in 6 females (19.4%; average age 50 ± 8). Herein, we demonstrated that the c.1901C>T variant is a loss-of-function splice-site variant that underlies the first CDH1-founder effect in Portugal. Knowledge on this founder effect will drive genetic testing of this specific variant in HDGC families in this geographical region and allow intrafamilial penetrance analysis and better estimation of variant-associated tumour risks, disease age of onset, and spectrum.

Integrative genome-wide analyses reveal the transcriptional aberrations in Japanese esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a malignant disease. At present, the genomic profiles of ESCC are known to a considerable extent, and DNA methylation and gene expression profiles have been mainly used for the classification of ESCC subtypes, but integrative genomic, transcriptomic, and epigenomic analyses remain insufficient. Therefore, we performed integrative analyses using whole‐exome sequencing, DNA methylation, and RNA sequencing (RNA‐seq) analyses of Japanese patients with ESCC. In cancer‐related genes, such as NOTCH family genes, RTK/PI3K pathway genes, and NFE2L2 pathway genes, variants and copy number amplification were detected frequently. Japanese ESCC cases were clustered into two mutational signatures: an APOBEC‐associated signature and an age‐related signature. In imprinted genes, DNA methylation was aberrant in gene promoter regions and correlated well with gene expression profiles. Nonsynonymous single‐nucleotide variants and allelic expression imbalance were detected frequently in FAT family genes. Our integrative genome‐wide analyses, including DNA methylation and allele‐specific gene expression profiles, revealed altered gene regulation of imprinted genes and FAT family genes in ESCC.

Perspectives on Allele-Specific Expression

Diploidy has profound implications for population genetics and susceptibility to genetic diseases. Although two copies are present for most genes in the human genome, they are not necessarily both active or active at the same level in a given individual. Genomic imprinting, resulting in exclusive or biased expression in favor of the allele of paternal or maternal origin, is now believed to affect hundreds of human genes. A far greater number of genes display unequal expression of gene copies due to cis-acting genetic variants that perturb gene expression. The availability of data generated by RNA sequencing applied to large numbers of individuals and tissue types has generated unprecedented opportunities to assess the contribution of genetic variation to allelic imbalance in gene expression. Here we review the insights gained through the analysis of these data about the extent of the genetic contribution to allelic expression imbalance, the tools and statistical models for gene expression imbalance, and what the results obtained reveal about the contribution of genetic variants that alter gene expression to complex human diseases and phenotypes.

Allelic expression imbalance refined genome-wide association signals in osteoarthritis

Purpose: A continuum of genome wide association studies (GWAS) on osteoarthritis (OA) with ever increasing sample sizes have resulted in 96 independent loci associating to OA related phenotypes. Consequently, the urge to annotate transcriptional effects of identified risk SNPs in OA relevant tissue is growing. To address functionality of identified risk SNPs we performed allelic expression imbalance (AEI) in whole transcriptomics data of articular cartilage from OA patients.