Multiple Gene Loci Research Articles

A novel methylation-detection panel for HPV associated high-grade squamous intraepithelial lesion and cervical cancer screening

Cervical cancer (CC) was considered to be the most common gynaecological cancer, with an estimated 342,000 deaths worldwide each year, as the majority of patients were diagnosed at an advanced stage of the disease. The purpose of this study was to evaluate the predictive value of multi-locus methylation assay for the early detection of CC. The cervical exfoliated cell samples from 492 HPV-positive females with cervical lesions were collected and subjected to methylation detection of gene FAM19A4, EPB41L3 and PAX1 after bisulfite conversion. The levels of gene methylation in patients with different severity of cervical lesions were evaluated and compared. The receiver-operating characteristic (ROC) curve was established and efficacy indexes such as sensitivity, specificity and area under the curve (AUC) were calculated to assess the diagnostic value of DNA methylation detection at multiple gene loci for CC. The methylation levels of FAM19A4, EPB41L3 and PAX1 were significantly increased with the grade of cervical squamous intraepithelial lesions. The sensitivities of FAM19A4, EPB41L3 and PAX1 alone for high-grade squamous intraepithelial lesion (HSIL) and CC diagnosis were 84.6%, 86.3% and 88.0%, respectively; when three markers were combined by a logistic regression model, the sensitivity was 88.0%, with a high specificity of 97.7% and AUC of 0.957 (95% CI 0.937–0.977). Methylation status of FAM19A4, EPB41L3 and PAX1 were highly specific and effective for monitoring the progression of cervical lesions and the tri-gene methylation assay could be used as a triage tool for CC early screening.

Predation of anurans in southern England by Batracobdella algira, a leech previously unknown in the UK

Leech predation of amphibians is known to occur in Europe. Observation of severe leech infestation affecting a common toad Bufo bufo in southern England in summer 2020, with leeches covering the toad’s eyes, throat and axillae, initiated a collaborative investigation to learn more about the occurrence of such leech predation of anurans in the UK. Soliciting reports from the general public identified leech predation of common toads and common frogs Rana temporaria in Devon, Greater London, Hampshire, the Isle of Wight and Somerset in southern England. Through morphological and/or molecular investigation of samples, Batracobdella algira, a leech species not previously reported in the UK, was identified in the majority of cases. The known native Placobdella costata was also identified, with the observed feeding behaviour on anuran hosts being indistinguishable from that of B. algira. Whether the latter is a previously unrecorded or an introduced species has not yet been established. However, sequence data from multiple gene loci were identical to B. algira found in Tunisia, suggesting it more likely to be a non-native species to the UK. Further work is required to elucidate the potential origin and distribution of B. algira in the UK and whether it has any impact on amphibian populations. Keywords: amphibians, Bufo bufo, Glossiphoniidae, Hirudinea, Rana temporaria

Modulation of the microhomology-mediated end joining pathway suppresses large deletions and enhances homology-directed repair following CRISPR-Cas9-induced DNA breaks

BackgroundCRISPR-Cas9 genome editing often induces unintended, large genomic rearrangements, posing potential safety risks. However, there are no methods for mitigating these risks.ResultsUsing long-read individual-molecule sequencing (IDMseq), we found the microhomology-mediated end joining (MMEJ) DNA repair pathway plays a predominant role in Cas9-induced large deletions (LDs). We targeted MMEJ-associated genes genetically and/or pharmacologically and analyzed Cas9-induced LDs at multiple gene loci using flow cytometry and long-read sequencing. Reducing POLQ levels or activity significantly decreases LDs, while depleting or overexpressing RPA increases or reduces LD frequency, respectively. Interestingly, small-molecule inhibition of POLQ and delivery of recombinant RPA proteins also dramatically promote homology-directed repair (HDR) at multiple disease-relevant gene loci in human pluripotent stem cells and hematopoietic progenitor cells.ConclusionsOur findings reveal the contrasting roles of RPA and POLQ in Cas9-induced LD and HDR, suggesting new strategies for safer and more precise genome editing.

Multilocus pathogenic variants contribute to intrafamilial clinical heterogeneity: a retrospective study of sibling pairs with neurodevelopmental disorders

BackgroundMultilocus pathogenic variants (MPVs) are genetic changes that affect multiple gene loci or regions of the genome, collectively leading to multiple molecular diagnoses. MPVs may also contribute to intrafamilial phenotypic variability between affected individuals within a nuclear family. In this study, we aim to gain further insights into the influence of MPVs on a disease manifestation in individual research subjects and explore the complexities of the human genome within a familial context.MethodsWe conducted a systematic reanalysis of exome sequencing data and runs of homozygosity (ROH) regions of 47 sibling pairs previously diagnosed with various neurodevelopmental disorders (NDD).ResultsWe found siblings with MPVs driven by long ROH regions in 8.5% of families (4/47). The patients with MPVs exhibited significantly higher FROH values (p-value = 1.4e-2) and larger total ROH length (p-value = 1.8e-2). Long ROH regions mainly contribute to this pattern; the siblings with MPVs have a larger total size of long ROH regions than their siblings in all families (p-value = 6.9e-3). Whereas the short ROH regions in the siblings with MPVs are lower in total size compared to their sibling pairs with single locus pathogenic variants (p-value = 0.029), and there are no statistically significant differences in medium ROH regions between sibling pairs (p-value = 0.52).ConclusionThis study sheds light on the significance of considering MPVs in families with affected sibling pairs and the role of ROH as an adjuvant tool in explaining clinical variability within families. Identifying individuals carrying MPVs may have implications for disease management, identification of possible disease risks to different family members, genetic counseling and exploring personalized treatment approaches.

An Emerging Role for Enhancer RNAs in Brain Disorders.

Noncoding DNA undergoes widespread context-dependent transcription to produce noncoding RNAs. In recent decades, tremendous advances in genomics and transcriptomics have revealed important regulatory roles for noncoding DNA elements and the RNAs that they produce. Enhancers are one such element that are well-established drivers of gene expression changes in response to a variety of factors such as external stimuli, cellular responses, developmental cues, and disease states. They are known to act at long distances, interact with multiple target gene loci simultaneously, synergize with other enhancers, and associate with dynamic chromatin architectures to form a complex regulatory network. Recent advances in enhancer biology have revealed that upon activation, enhancers transcribe long noncoding RNAs, known as enhancer RNAs (eRNAs), that have been shown to play important roles in enhancer-mediated gene regulation and chromatin-modifying activities. In the brain, enhancer dysregulation and eRNA transcription has been reported in numerous disorders from acute injuries to chronic neurodegeneration. Because this is an emerging area, a comprehensive understanding of eRNA function has not yet been achieved in brain disorders; however, the findings to date have illuminated a role for eRNAs in activity-driven gene expression and phenotypic outcomes. In this review, we highlight the breadth of the current literature on eRNA biology in brain health and disease and discuss the challenges as well as focus areas and strategies for future in-depth research on eRNAs in brain health and disease.

Mechanisms of action and resistance in histone methylation-targeted therapy

Epigenomes enable the rectification of disordered cancer gene expression, thereby providing new targets for pharmacological interventions. The clinical utility of targeting histone H3 lysine trimethylation (H3K27me3) as an epigenetic hallmark has been demonstrated1–7. However, in actual therapeutic settings, the mechanism by which H3K27me3-targeting therapies exert their effects and the response of tumour cells remain unclear. Here we show the potency and mechanisms of action and resistance of the EZH1–EZH2 dual inhibitor valemetostat in clinical trials of patients with adult T cell leukaemia/lymphoma. Administration of valemetostat reduced tumour size and demonstrated durable clinical response in aggressive lymphomas with multiple genetic mutations. Integrative single-cell analyses showed that valemetostat abolishes the highly condensed chromatin structure formed by the plastic H3K27me3 and neutralizes multiple gene loci, including tumour suppressor genes. Nevertheless, subsequent long-term treatment encounters the emergence of resistant clones with reconstructed aggregate chromatin that closely resemble the pre-dose state. Acquired mutations at the PRC2–compound interface result in the propagation of clones with increased H3K27me3 expression. In patients free of PRC2 mutations, TET2 mutation or elevated DNMT3A expression causes similar chromatin recondensation through de novo DNA methylation in the H3K27me3-associated regions. We identified subpopulations with distinct metabolic and gene translation characteristics implicated in primary susceptibility until the acquisition of the heritable (epi)mutations. Targeting epigenetic drivers and chromatin homeostasis may provide opportunities for further sustained epigenetic cancer therapies.

Needle pathogens of Rhytismataceae: Current knowledge and research opportunities for conifer foliar diseases

AbstractNeedle diseases caused by pathogens within Rhytismataceae are increasing in frequency globally where pines are either a predominate species or are grown commercially, and yet remain poorly understood. Here, we provide background information on the current knowledge and management strategies of needle pathogens in the Rhytismataceae and discuss research opportunities using molecular tools to fill knowledge gaps. Many of the aggressive needle pathogens within this group lack morphological features to delimit species and are difficult to grow in axenic cultures, which complicates scientific investigations. Molecular research has greatly advanced knowledge of this unique group of pathogens. Nucleotide sequences from multiple gene loci identified greater diversity within this group of pathogens than previously thought, as new and cryptic species are being discovered in broader geographic regions. High throughput sequencing and analyses revealed that an endophytic lifestyle is a common trait among fastidious if not obligate needle pathogens within Rhytismataceae. Further, molecular analyses showed the impact these pathogens have on the diversity of endophytic microbiota is underestimated. Early detection in healthy‐looking needles using PCR‐based markers greatly improves capabilities for disease diagnosis and monitoring. Despite growing number of molecular studies on Rhytismataceae needle pathogens, fundamental questions on the taxonomy, phylogeny, ecology and pathology of many Rhytismataceae needle pathogens remain unanswered. Nonetheless, with the projected changes in the occurrence and severity of Rhytismataceae pathogens, increased accessibility to molecular tools vastly expands the potential to gain more insights into understudied needle pathosystems to help define and address its impact under changing environments and consequently inform forest disease management more effectively.

Statistical Study Design for Analyzing Multiple Gene Loci Correlation in DNA Sequences

This study presents a novel statistical and computational approach using nonparametric regression, which capitalizes on correlation structure to deal with the high-dimensional data often found in pharmacogenomics, for instance, in Crohn’s inflammatory bowel disease. The empirical correlation between the test statistics, investigated via simulation, can be used as an estimate of noise. The theoretical distribution of −log10(p-value) is used to support the estimation of that optimal bandwidth for the model, which adequately controls type I error rates while maintaining reasonable power. Two proposed approaches, involving normal and Laplace-LD kernels, were evaluated by conducting a case-control study using real data from a genome-wide association study on Crohn’s disease. The study successfully identified single nucleotide polymorphisms on the NOD2 gene associated with the disease. The proposed method reduces the computational burden by approximately 33% with reasonable power, allowing for a more efficient and accurate analysis of genetic variants influencing drug responses. The study contributes to the advancement of statistical methodology for analyzing complex genetic data and is of practical advantage for the development of personalized medicine.

New evidence of consistency between phylogeny and morphology for two taxa in ciliated protists, the subclasses Oligotrichia and Choreotrichia (Protista, Ciliophora)

Marine planktonic ciliates are largely oligotrichs and choreotrichs, which are two subclasses of the class Spirotrichea. The current phylogenetic assignments of oligotrichs and choreotrichs are inconsistent with previous results based on morphological features, probably hindered by the limited information from a single gene locus. Here we provide 53 new sequences from small subunit ribosomal RNA (SSU rDNA), ITS1-5.8S rDNA-ITS2, and large subunit ribosomal RNA (LSU rDNA) gene loci in 25 oligotrich and choreotrich species. We also predict RNA secondary structures for the ITS2 regions in 55 species, 48 species of which are reported for the first time. Based on these novel data, we make a more comprehensive phylogenetic reconstruction, revealing consistency between morphological taxonomy and an updated phylogenetic system for oligotrichs and choreotrichs. With the addition of data from ciliature patterns and genes, the phylogenetic analysis of the subclass Oligotrichia suggests three evolutionary trajectories, among which: 1) Novistrombidium asserts an ancestral ciliary pattern in Oligotrichia; 2) the subgenera division of Novistrombidium and Parallelostrombidium are fully supported; 3) the three families (Tontoniidae, Pelagostrombidiidae and Cyrtostrombidiidae) all evolved from the most diverse family Strombidiidae, which explains why strombidiids consistently form polyphyletic clades. In the subclass Choreotrichia, Strombidinopsis likely possesses an ancestral position to other choreotrichs, and both phylogenetic analysis and RNA secondary structure prediction support the hypothesis that tintinnids may have evolved from Strombidinopsis. The results presented here offer an updated hypothesis for the evolutionary history of oligotrichs and choreotrichs based on new evidence obtained by expanding sampling of molecular information across multiple gene loci.

Morphological and molecular characteristics of a single oocyst for the identification of Eimeria species in domestic rabbits (Oryctolagus cuniculus f. domesticus)

Coccidiosis caused by Eimeria is one of the most common diseases in domestic rabbits (Oryctolagus cuniculus f. domesticus), with 11 Eimeria species in domestic rabbits recognized internationally. To identify Eimeria species more accurately, a method based on the molecular characteristics of a single oocyst with multiple gene loci was established by combining morphological and molecular biology. The results showed that the total infection rate of Eimeria in domestic rabbits was 44.2 % (152/344). Ten Eimeria species were identified in domestic rabbits based on morphological characteristics, namely Eimeria vejdovskyi (39.5 %, 136/344), E. magna (18.0 %, 62/344), E. perforans (17.4 %, 60/344), E. intestinalis (12.5 %, 43/344), E. media (11.9 %, 41/344), E. coecicola (4.4 %, 15/344), E. irresidua (3.8 %, 13/344), E. exigua (2.6 %, 9/344), E. stiedai (2.3 %, 8/344), and E. piriformis (1.5 %, 5/344). The molecular biological identification of Eimeria in domestic rabbits was conducted through single oocyst selection and nested polymerase chain reaction amplification with multiple gene loci. We obtained the sequences of the 18S rRNA, ITS-1 and COI gene loci of E. magna, E. perforans, E. vejdovskyi, E. media, E. intestinalis, and E. coecicola. The results showed that the molecular biology and morphological identification results of single oocysts were consistent and could be used for the molecular identification of Eimeria at the single oocyst level. This study provides an efficient tool for identification of Eimeria in domestic rabbits and the population genetic study of Eimeria in domestic rabbits.

Detection of DNA Methylation in Gene Loci ASTN1, DLX1, ITGA4, RXFP3, SOX17, and ZNF671 for Diagnosis of Cervical Cancer.

To evaluate the diagnostic value of DNA methylation detection of multiple gene loci in cervical cancer. A total of 61 cases requiring cervical biopsy were selected from the outpatient clinic of Maternal and Child Health Hospital of Hubei Province between January 2018 and December 2019. The patients were divided into four groups based on histopathologic diagnosis: cervical cancer (CC) group, high-grade squamous intraepithelial lesion (HSIL) group, low-grade squamous intraepithelial lesion (LSIL) group, and control group. HPV examination, liquid-based cytology examination, and DNA methylation detection at multiple gene sites were performed. The positive rate of DNA methylation, sensitivity, specificity, area under the curve (AUC), and other efficacy indexes were calculated to evaluate the diagnostic value of DNA methylation detection at multiple gene loci in cervical cancer. The positive rates of DNA methylation in CC, HSIL, LSIL, and control groups were 100%, 88%, 83% and 17%, respectively. The ZNF671 gene had the highest positive rate among the cervical lesion group, with rates of 57%, 76%, and 100% in LSIL, HSIL, and CC groups respectively. The combination of DNA methylation detection at multiple gene loci showed the highest diagnostic efficacy for HSIL and cervical cancer, with AUC value of 0.850 (95% CI:0.746-0.954), a Youden index of 0.654, and a sensitivity and specificity of 85% and 85.4%, respectively. The diagnostic efficacy of the combined detection was significantly higher than that of HPV examination and liquid-based cytology examination (P < 0.05). DNA methylation detection at multiple gene loci is highly effective and diagnostic tool for cervical cancer, and has potential application value in clinical practice.

BH14 Multiple sclerosis and alopecia areata: drug-induced alopecia areata or coexisting autoimmune phenomena?

Abstract Alopecia areata (AA) is an autoimmune condition characterized by well-demarcated patches of nonscarring alopecia. AA shares multiple gene loci with other autoimmune conditions, including multiple sclerosis (MS). There are limited data on drug-induced alopecia areata (DIAA); however, the literature is expanding, with several targeted therapies implicated including dupilumab, nivolumab and multiple disease-modifying MS treatments, namely, alemtuzumab and ocrelizumab. Alemtuzumab is monoclonal antibody against CD52, which is expressed on T and B lymphocytes, natural killer (NK) cells, monocytes and macrophages. It is considered a highly efficacious treatment; however, there are increasing reports of secondary autoimmunity in up to 40% of patients (Tuohy O, Costellow L, Hill-Cawthorne G et al. Alemtuzumab treatment of MS: long-term safety and efficacy. J Neurol Neurosurg Psychiatry 2015; 86:208–15). A 35-year-old man was referred by his neurologist with concerns of alemtuzumab-induced alopecia universalis (AU). He has relapsing-remitting MS, diagnosed in 2018. His first alemtuzumab course was in 2018 and the second in June 2019, which achieved remission. He reported AU approximately 10 months after this second course with complete spontaneous regrowth. A further episode of AU occurred 6 months later with only minimal eyebrow regrowth. He had no previous history of alopecia or thyroid disease. He had vitamin D insufficiency. There are 12 cases of alemtuzumab-induced AA reported in the literature with variable timeframes of onset, ranging between weeks to over 6–9 months postinfusion. The majority of patients received more than one infusion prior to the onset of AA. Our case echoes other case reports, including one case of AU manifesting 9 months post-alemtuzumab (Zimmermann J. Buhl T, Muller M. AU following alemtuzumab treatment in MS: a barely recognized manifestation of secondary autoimmunity – case report and review of literature. Front Neurol 2017; 8:569). AA pathogenesis, like MS, is hypothesized to be multifactorial with genetic, environmental and autoimmune aspects. It is believed that AA hair follicle cells have increased major histocompatibility complex class I and II activity, presenting autoantigens to CD8+ T cells, CD4+ T cells and NK cells. Interferon (IFN)-γ is considered key in AA pathogenesis. Patients with AA are found to have raised serum levels, along with reports of raised interleukin (IL)-6 levels. Alemtuzumab-induced immunosuppression precedes a phase of immune reconstitution. Cytokines are released post-treatment, including IFN-γ, tumour necrosis factor-α, IL-6 and IL-2. It is plausible that alemtuzumab could trigger AA as a sequela of the immune reconstitution. However, DIAA has been poorly described and the genetic overlap raises the question of both conditions occurring spontaneously. Accurate phenotyping and a multidisciplinary approach will enable us to gain greater clarity, inform our decision-making in the management of these complex patients and provide insights into pathogenic AA pathways.

On the composition of

Morphological and molecular studies have consistently suggested that Sminthopsis, as currently defined, is rendered paraphyletic by the kultarr (Antechinomys laniger). They have also suggested a sister relationship between the kultarr and the long-tailed dunnart. Based on DNA sequence data from multiple mitochondrial and nuclear gene loci we reassign the long-tailed dunnart (formerly Sminthopsis longicaudata) to Antechinomys. Although there is good evidence of genetic structure within the kultarr (A. laniger), it does not correspond to the two currently recognised subspecies, viz A. laniger laniger and A. l. spenceri. We conclude that Antechinomys consists of two species, A. laniger and A. longicaudatus, consistent with morphology. We suggest that the observed genetic and morphological variation within A. laniger merits a more thorough investigation of more samples from across its range to resolve the taxonomy.

Durable Clinical Impacts and Mechanisms of Action and Resistance in EZH1/2-Targeting Epigenetic Therapy

Background: Epigenomes allow the rectification of disordered gene expressions, thereby providing new targets for pharmacological interventions in cancer therapy. The clinical utility of targeting histone H3 lysine tri-methylation (H3K27me3) as an epigenetic hallmark has been demonstrated. However, in actual therapeutic settings, the mechanism by which H3K27me3-targeting therapies exert their effects and the response of tumor cells remain unclear. Results and Discussion: We conducted intensive molecular monitoring of three patients enrolled in the valemetostat first-in-human phase 1 clinical trial (NCT02732275) for adult T-cell leukemia/lymphoma (ATL). Valemetostat was administered orally once daily (200 mg/day) until a sign of disease progression could be observed. The number of abnormal lymphocytes (Ably) in these three patients drastically decreased after one week of treatment. All patients could stay on valemetostat single agent for more than 2 years with acceptable safety and durable response. PBMC samples (n = 55) were collected from these three patients immediately before (Pre) and during treatment, as well as during the progressive disease (PD) stage. Flow cytometry-based histone methylation assay demonstrated that tumor H3K27me3 level was generally high in the Pre-samples, but the valemetostat treatment reduced it to a normal level in all patients. In addition, ChIP sequencing (ChIP-seq) could directly assess how valemetostat affects the tumor epigenome. The pre-treatment tumor cells showed an overall increasing trend in H3K27me3. For all H3K27me3 peaks (51,683 peaks), 85.9% of the peak signals decreased at the average fold-change of 0.742 after the valemetostat treatment. Focusing on the gene loci, an overall decrease could be observed around the TSS and across the gene body. The characteristic H3K27me3 clusters initially observed in the tumor cells were demethylated. We also conducted integrative multilayered single-cell analyses (scATAC-seq and scRNA-seq) and showed that valemetostat abolishes the highly condensed chromatin structure formed by the plastic H3K27me3 and neutralizes multiple gene loci, such as tumor suppressor genes (TSGs), in all patients. These results demonstrated that valemetostat sufficiently restored the epigenome of the tumor cells close to the healthy state, leading to clinical improvements. Although all tested cases showed a durable response for more than 2 years, these responses were eventually interrupted due to the recurrence of ATL. We identified characteristic somatic mutations of EZH2 during clonal repopulation at PD. The identified amino acid substitutions were all located at the interface between EZH2 and valemetostat. Direct evaluation of the H3K27me3 level in the PD sample harboring the acquired EZH2 mutation showed that tumor cells in a hypermethylated state were repopulating. The scATAC-seq analysis also showed strong chromatin compaction of the same gene set occurred in the rapidly propagating resistant clone, indicating that the clonally selected mutations should be responsible for resistance. In patients free of the EZH2 resistant mutations, we detected TET2 mutation or elevated DNMT3A expression in the propagated resistant clones. Genome-wide DNA methylation analysis revealed increased DNA methylation near the TSS in the resistant clones. The genes characterized by the H3K27me3-related chromatin condensation were restored by valemetostat treatment but were again strongly re-condensed by mCpG gain at PD. Consequently, the gene regulation of several important TSGs restored by valemetostat was repressed again. Furthermore, in the single-cell platform, we successfully identified subpopulations with distinct metabolic and gene translation characteristics implicated in primary susceptibility until the acquisition of the heritable (epi)mutations. Conclusion: This study illustrates, for the first time, the molecular and cellular dynamics in patients in response to a molecular-targeting inhibitor designed for histone methyltransferases. Eliminating H3K27me3 leads to the reprogramming of the cancer epigenome, thereby exerting a sustained benefit on tumors. Targeting chromatin homeostasis might provide opportunities for further sustained epigenetic therapies.

HSF1 Stimulates Glutamine Transport by Super-Enhancer-Driven lncRNA LINC00857 in Colorectal Cancer.

Simple SummaryBased on the latest research, cancer cells prefer glutamine to glucose. Therefore, it is more worthwhile to explore the regulatory mechanism of glutamine metabolism in cancer cells. Super enhancers are critical for the gene transcriptional programs responsible for cell fate by interacting with various transcription factors. The transcription factor HSF1 exerts a multifaced role in tumorigenesis. However, the relevance of HSF1 to super enhancers in tumors remains obscure. Therefore, this study focused on the mechanism underlying super-enhancer activation and its relationship to HSF1 in CRC. Here, we performed a super-enhancer landscape in CRC and we screened out an HSF1-mediated super enhancer, lncRNA-LINC00857, by lncRNA microarray. We discovered that HSF1 could stimulate acetyltransferase P300-mediated super-enhancer activity to facilitate LINC00857 expression, contributing to SLC1A5/ASCT2-mediated glutamine transport. In addition, we validated that targeting the HSF1/LINC00857/ANXA11 axis may provide a valuable therapeutic strategy against CRC.Super enhancers are critical for the gene transcription responsible for cell fate by interacting with transcription factors. However, the relevance of HSF1 to super enhancers in tumors remains obscure. We profiled H3K27ac enrichment by chromatin immunoprecipitation sequencing. HSF1-mediated lncRNAs were identified by lncRNA microarray. The characteristics of LINC00857 were explored by in vitro and in vivo assays. The mechanism was studied via chromatin immunoprecipitation, RNA immunoprecipitation, and HSF1/ANXA11 knockout mice. We found that super enhancers occupied multiple gene loci in colorectal cancer. We screened out an HSF1-mediated super enhancer, lncRNA-LINC00857, which exerts its characteristics in promoting cell growth via regulating glutamine metabolism. Notably, HSF1 could stimulate the super-enhancer activity of LINC00857 by the enrichment of acetyltransferase P300 to its gene loci, contributing to LINC00857 transcription. In turn, nuclear LINC00857 cooperated with HSF1 to promote ANXA11 transcription, which modulated SLC1A5/ASCT2 protein expression by binding competitively to miR-122-5p. The knockout of ANXA11 attenuated colorectal cancer formation in vivo. Collectively, we shed light on a closely cooperative machinery between HSF1 and super enhancers. HSF1 could stimulate acetyltransferase P300-mediated super-enhancer activity to facilitate LINC00857 expression, contributing to SLC1A5-mediated glutamine transport. Targeting the HSF1/LINC00857/ANXA11 axis may provide a valuable therapeutic strategy against colorectal cancer.

Sequence Variations Within HLA-G and HLA-F Genomic Segments at the Human Leukocyte Antigen Telomeric End Associated With Acute Graft-Versus-Host Disease in Unrelated Bone Marrow Transplantation.

Acute graft-versus-host disease (aGVHD) is defined as a syndrome of an immunological response of graft to the host that occurs early after allogeneic hematopoietic stem cell transplantation (HCT). This disease is frequently observed even in HCT matched for human leukocyte antigen (HLA) alleles at multiple gene loci. Although the HLA region represents complex and diverse genomic characteristics, detailed association analysis is required for the identification of uncharacterized variants that are strongly associated with aGVHD. We genotyped three loci, OR2H2, HLA-F-AS1, and HLA-G, that are located in the 460 kb of HLA telomeric region and statistically analyzed the genotypes including HLA-DPB1 with clinical and transplantation outcomes using 338 unrelated bone marrow transplantation (UR-BMT) patient–donor pairs who were matched for HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 (HLA-10/10). Multivariate analyses demonstrated that HLA-F-AS1 and HLA-DPB1 mismatches were associated with grade II–IV aGVHD (hazard ratio (HR), 1.76; 95% CI, 1.07–2.88; p = 0.026; and HR, 1.59; CI, 1.02–2.49; p = 0.042, respectively). There was no confounding between HLA-F-AS1 and HLA-DPB1 (p = 0.512), suggesting that the HLA-F-AS1 mismatch has a strong effect on aGVHD independently of HLA-DPB1. Moreover, a stratified analysis suggested possible associations of HLA-F-AS1, HLA-DPB1, and/or HLA-G mismatches with grade II–IV aGVHD and the more severe grade III–IV aGVHD. These findings provide new insights into understanding the molecular mechanism of aGVHD caused by HLA-matched UR-BMT.

Simulation Research on the Methods of Multi-Gene Region Association Analysis Based on a Functional Linear Model.

Genome-wide association analysis is an important approach to identify genetic variants associated with complex traits. Complex traits are not only affected by single gene loci, but also by the interaction of multiple gene loci. Studies of association between gene regions and quantitative traits are of great significance in revealing the genetic mechanism of biological development. There have been a lot of studies on single-gene region association analysis, but the application of functional linear models in multi-gene region association analysis is still less. In this paper, a functional multi-gene region association analysis test method is proposed based on the functional linear model. From the three directions of common multi-gene region method, multi-gene region weighted method and multi-gene region loci weighted method, that test method is studied combined with computer simulation. The following conclusions are obtained through computer simulation: (a) The functional multi-gene region association analysis test method has higher power than the functional single gene region association analysis test method; (b) The functional multi-gene region weighted method performs better than the common functional multi-gene region method; (c) the functional multi-gene region loci weighted method is the best method for association analysis on three directions of the common multi-gene region method; (d) the performance of the Step method and Multi-gene region loci weighted Step for multi-gene regions is the best in general. Functional multi-gene region association analysis test method can theoretically provide a feasible method for the study of complex traits affected by multiple genes.

Genomic compatibility and inheritance of hexaploid-derived Fusarium head blight resistance genes in durum wheat.

Hexaploid-derived resistance genes exhibit complex inheritance and expression patterns in tetraploid backgrounds. This study aimed to characterize the inheritance patterns and genomic compatibilities of hexaploid-derived Fusarium head blight (FHB) resistance genes in tetraploid durum wheat (Triticum durumDesf.). Evaluation of FHB resistance for F1 hybrids of hexaploid 'Sumai 3' crossed with tetraploid and hexaploid wheats indicated that Sumai 3-derived FHB resistance genes exhibit a dominant phenotypic effect seen only in hexaploid hybrids. Alternately, the hexaploid-derived FHB resistance genes from PI 277012 exhibited complete dominance in the crosses with both tetraploid and hexaploid wheat. FHB evaluation of the F1 hybrids of Sumai 3 and PI 277012 crossed with 'Langdon' (LDN)-'Chinese Spring' D-genome substitution lines suggested that chromosomes 2B, 3B, 4B, 5B, 6B, 3A, 4A, 6A, and 7A contain genes that suppress expression of the Sumai 3-derived FHB resistance, whereas chromosomes 4A, 6A, and 6B contain genes required for expression of PI 277012-derived FHB resistance. A wide range of segregation for FHB severity (10-90%) was observed in the F2 generation of Sumai 3 crossed with durum cultivars LDN and 'Divide', but the distribution of F3 families derived from the most resistant F2 segregants was skewed towards susceptibility. Similar segregation trends were observed in the crosses of PI 277012 with other durum wheats, whereby FHB resistance became slightly diluted over successive generations. These results suggest tetraploid durum wheat contains the unique alleles at multiple gene loci on different chromosomes that positively and/or negatively regulate the expression of hexaploid-derived FHB resistance genes, which complicate efforts to deploy these genes in durum breeding programs.

Adenine base-editing-mediated exon skipping induces gene knockout in cultured pig cells.

Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9, Adenine base editor (ABE) convert single A·T pairs to G·C pairs in the genome without generating DNA double-strand breaks, and this method has higher accuracy and biosafety in pig genetic modification. However, the application of ABE in pig gene knockout is limited by protospacer-adjacent motif sequences and the base-editing window. Alternative mRNA splicing is an important mechanism underlying the formation of proteins with diverse functions in eukaryotes. Spliceosome recognizes the conservative sequences of splice donors and acceptors in a precursor mRNA. Mutations in these conservative sequences induce exon skipping, leading to proteins with novel functions or to gene inactivation due to frameshift mutations. In this study, adenine base-editing-mediated exon skipping was used to expand the application of ABE in the generation of gene knockout pigs. We first constructed a modified "all-in-one" ABE vector suitable for porcine somatic cell transfection that contained an ABE for single-base editing and an sgRNA expression cassette. The "all-in-one" ABE vector induced efficient sgRNA-dependent A-to-G conversions in porcine cells during single base-editing of multiple endogenous gene loci. Subsequently, an ABE system was designed for single adenine editing of the conservative splice acceptor site (AG sequence at the 3' end of the intron 5) and splice donor site (GT sequence at the 5' end of the intron 6) in the porcine gene GHR; this method achieved highly efficient A-to-G conversion at the cellular level. Then, porcine single-cell colonies carrying a biallelic A-to-G conversion in the splice acceptor site in the intron 5 of GHR were generated. RT-PCR indicated exon 6 skipped at the mRNA level. Western blotting revealed GHR protein loss, and gene sequencing showed no sgRNA-dependent off-target effects. These results demonstrate accurate adenine base-editing-mediated exon skipping and gene knockout in porcine cells. This is the first proof-of-concept study of adenine base-editing-mediated exon skipping for gene regulation in pigs, and this work provides a new strategy for accurate and safe genetic modification of pigs for agricultural and medical applications.

Diagnosis of Sickle Cell Disease and HBB Haplotyping in the Era of Personalized Medicine: Role of Next Generation Sequencing.

Hemoglobin genotype and HBB haplotype are established genetic factors that modify the clinical phenotype in sickle cell disease (SCD). Current methods of establishing these two factors are cumbersome and/or prone to errors. The throughput capability of next generation sequencing (NGS) makes it ideal for simultaneous interrogation of the many genes of interest in SCD. This study was designed to confirm the diagnosis in patients with HbSS and Sβ-thalassemia, identify any ß-thal mutations and simultaneously determine the ßS HBB haplotype. Illumina Ampliseq custom DNA panel was used to genotype the DNA samples. Haplotyping was based on the alleles on five haplotype-specific SNPs. The patients studied included 159 HbSS patients and 68 Sβ-thal patients, previously diagnosed using high performance liquid chromatography (HPLC). There was considerable discordance between HPLC and NGS results, giving a false +ve rate of 20.5% with a sensitivity of 79% for the identification of Sβthal. Arab/India haplotype was found in 81.5% of βS chromosomes, while the two most common, of the 13 β-thal mutations detected, were IVS-1 del25 and IVS-II-1 (G>A). NGS is very versatile and can be deployed to simultaneously screen multiple gene loci for modifying polymorphisms, to afford personalized, evidence-based counselling and early intervention.