The Genetic Research in Alzheimer Disease (GERALD) Initiative Finds rs9320913 as a Neural eQTL of lincRNA AL589740.1

Alzheimer’s disease is the most common cause of dementia worldwide, and longitudinal studies are crucial to find the factors affecting disease development. Here, we describe a novel initiative from southern Spain designed to contribute in the identification of the genetic component of the cognitive decline of Alzheimer’s disease patients. The germline variant rs9320913 is a C>A substitution mapping within a gene desert. Although it has been previously associated to a higher educational achievement and increased fluid intelligence, its role on Alzheimer’s disease risk and progression remains elusive. A total of 407 subjects were included in the study, comprising 153 Alzheimer disease patients and 254 healthy controls. We have explored the rs9320913 contribution to both Alzheimer disease risk and progression according to the Mini-Mental State Exams. We found that rs9320913 maps within a central nervous system lincRNA AL589740.1. eQTL results show that rs9320913 correlated with the brain-frontal cortex ( beta = − 0.15 , p value = 0.057) and brain-spinal cord (beta of -0.23, p value = 0.037). We did not find rs9320913 to be associated to AD risk, although AA patients seemed to exhibit a less pronounced Mini-Mental State Exam score decline.

Identification of Novel Metabolism-Associated Subtypes for Pancreatic Cancer to Establish an Eighteen-Gene Risk Prediction Model

Pancreatic cancer (PanC) is an intractable malignancy with a high mortality. Metabolic processes contribute to cancer progression and therapeutic responses, and histopathological subtypes are insufficient for determining prognosis and treatment strategies. In this study, PanC subtypes based on metabolism-related genes were identified and further utilized to construct a prognostic model. Using a cohort of 171 patients from The Cancer Genome Atlas (TCGA) database, transcriptome data, simple nucleotide variants (SNV), and clinical information were analyzed. We divided patients with PanC into metabolic gene-enriched and metabolic gene-desert subtypes. The metabolic gene-enriched subgroup is a high-risk subtype with worse outcomes and a higher frequency of SNVs, especially in KRAS. After further characterizing the subtypes, we constructed a risk score algorithm involving multiple genes (i.e., NEU2, GMPS, PRIM2, PNPT1, LDHA, INPP4B, DPYD, PYGL, CA12, DHRS9, SULT1E1, ENPP2, PDE1C, TPH1, CHST12, POLR3GL, DNMT3A, and PGS1). We verified the reproducibility and reliability of the risk score using three validation cohorts (i.e., independent datasets from TCGA, Gene Expression Omnibus, and Ensemble databases). Finally, drug prediction was completed using a ridge regression model, yielding nine candidate drugs for high-risk patients. These findings support the classification of PanC into two metabolic subtypes and further suggest that the metabolic gene-enriched subgroup is associated with worse outcomes. The newly established risk model for prognosis and therapeutic responses may improve outcomes in patients with PanC.

Role of lncRNA BANCR in Human Cancers: An Updated Review

Being located in a gene desert region on 9q21.11-q21.12, BRAF-activated non-protein coding RNA (BANCR) is an lncRNA with 693 bp length. It has been discovered in 2012 in a research aimed at assessment of gene expression in the melanocytes in association with BRAF mutation. Increasing numbers of studies have determined its importance in the tumorigenesis through affecting cell proliferation, migration, invasion, apoptosis, and epithelial to mesenchymal transition. BANCR exerts its effects via modulating some tumor-related signaling pathways particularly MAPK and other regulatory mechanisms such as sponging miRNAs. BANCR has been up-regulated in endometrial, gastric, breast, melanoma, and retinoblastoma. Conversely, it has been down-regulated in some other cancers such as those originated from lung, bladder, and renal tissues. In some cancer types such as colorectal cancer, hepatocellular carcinoma and papillary thyroid carcinoma, there is no agreement about BANCR expression, necessitating the importance of additional functional studies in these tissues. In the present manuscript, we review the investigations related to BANCR expression changes in cancerous cell lines, clinical samples, and animal models of cancer. We also discuss the outcome of its deregulation in cancer progression, prognosis, and the underlying mechanisms of these observations.

BCL11B Regulates Arterial Stiffness and Related Target Organ Damage

Rationale: B-cell leukemia 11b (BCL11B) is a transcription factor known as an essential regulator of T lymphocytes and neuronal development during embryogenesis. A genome-wide association study (GWAS) showed that a gene desert region downstream of BCL11B, known to function as a BCL11B enhancer, harbors single nucleotide polymorphisms (SNPs) associated with increased arterial stiffness. However, a role for BCL11B in the adult cardiovascular system is unknown. Objective: Based on these human findings, we sought to examine the relation between BCL11B and arterial function. Methods and Results: Here we report that BCL11B is expressed in the vascular smooth muscle (VSM) where it regulates vascular stiffness. RNA sequencing of aortas from WT and Bcl11b null mice (BSMKO) identified the cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) as the most significant differentially regulated signaling pathway in BSMKO compared to WT mice. BSMKO aortas showed decreased levels of PKG1, increased levels of Ca ++ -calmodulin-dependent serine/threonine phosphatase calcineurin (PP2B) and decreased levels of their common phosphorylation target, vasodilator-stimulated phosphoprotein (pVASP S239 ), a regulator of cytoskeletal actin rearrangements. Decreased pVASP S239 in BSMKO aortas was associated with increased actin polymerization (F/G actin ratio). Functionally, aortic force, stress, wall tension and stiffness, measured ex vivo in organ baths, were increased in BSMKO aortas, and BSMKO mice had increased pulse wave velocity, the in vivo index of arterial stiffness. Despite having no effect on blood pressure or microalbuminuria, increased arterial stiffness in BSMKO mice was associated with increased incidence of cerebral microbleeds compared to age-matched WT littermates. Conclusions: We have identified VSM BCL11B as a crucial regulator of aortic smooth muscle function and a potential therapeutic target for vascular stiffness.

A gene desert required for regulatory control of pleiotropic Shox2 expression and embryonic survival

ABSTRACTThe Shox2 homeodomain transcriptional regulator is known for its critical functions during mouse embryogenesis, enabling accurate development of limbs, craniofacial structures, neural populations and the cardiac conduction system. At the genomic level, the Shox2 gene is flanked by an extensive gene desert, a continuous non-coding genomic region spanning over 500 kilobases that contains a multitude of evolutionarily conserved elements with predicted cis-regulatory activities. However, the transcriptional enhancer potential of the vast majority of these elements in combination with the biological necessity of the gene desert have not yet been explored. Using transgenic reporter assays in mouse embryos to validate an extensive set of stringent epigenomic enhancer predictions, we identify several novel gene desert enhancers with distinct tissue-specific activities in Shox2 expressing tissues. 4C-seq chromatin conformation capture further uncovers a repertoire of gene desert enhancers with overlapping activities in the proximal limb, in a compartment essential for Shox2-mediated stylopod formation. Leveraging CRISPR/Cas9 to delete the gene desert region contained in the Shox2 topologically associated domain (TAD), we demonstrate that this complex cis-regulatory platform is essential for embryonic survival and required for control of region-specific Shox2 expression in multiple developing tissues. While transcription of Shox2 in the embryonic limb is only moderately affected by gene desert loss, Shox2 expression in craniofacial and cardiac domains is nearly abolished. In particular, Shox2 transcripts in the sinus venosus (SV) encompassing the sinoatrial node (SAN) were depleted in embryos lacking the gene desert, likely accounting for the embryonic lethality due to Shox2-dependency of the SAN pacemaker. Finally, we discover a 1.5kb SV enhancer within the deleted gene desert region, which may act as a genomic module controlling the development of the cardiac conduction system. In summary, our results identify a gene desert indispensable for pleiotropic patterning and highlight the importance of these extensive regulatory landscapes for embryonic development and viability.

Molecular mechanism of a new cardiac syndrome associated with a regulatory element deletion of chromosome 4q25

Introduction We identified a large family of 53 members of whom 22 present a new cardiac syndrome characterized by electrical disorders and developmental defects following an autosomal dominant model. Among the affected family members 6 are implanted with a pacemaker, 2 experienced syncope and one a sudden death at 43yo. Linkage analysis points, with high confidence, to the chromosome 4q25 region. This region is associated with the Ankyrin syndrome (mutation in ANK2) sharing partly the electrical defects observed in the affected family members. No mutation was found in the coding region of the 4q25 region as well as in the coding and non-coding part of the ANK2. Objective Our aims are first to identity the responsible mutation present in this family and understand the molecular mechanisms leading to this new syndrome. Method Whole genome sequencing (WGS) has been employed to identify genetic variants responsible for this syndrome. ChIP-seq and ATAC-seq were used for functional annotation and genome editing (CRISPR-Cas9) to generate iPS cellular models. Results By WGS we uncovered a deleted region of 15kb in a gene desert area on 4q25, segregating in all affected relatives. Five other families (3 French and 2 Japanese) presenting the same phenotype show overlapping deletions. We generated human cardiac epigenetic data and identified among the 15kb deleted region a unique active enhancer region within the presence of a transcript factor CTCF binding site. Isogenic cell lines where the 15kb and the CTCF binding have been deleted are under investigation. Conclusion We identified a new cardiac syndrome and for the first time a mutation located within a gene desert area leading to severe and complex cardiac disorders. We demonstrated the presence of a likely gene regulatory element. Experiments are ongoing to characterize the molecular mechanisms and consequence of the deletion on gene expression. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): Pays de la loire - Etoiles montantes

Trait-associated noncoding variant regions affect TBX3 regulation and cardiac conduction

Genome-wide association studies have implicated common genomic variants in the gene desert upstream of TBX3 in cardiac conduction velocity. Whether these noncoding variants affect expression of TBX3 or neighboring genes and how they affect cardiac conduction is not understood. Here, we use high-throughput STARR-seq to test the entire 1.3 Mb human and mouse TBX3 locus, including two cardiac conduction-associated variant regions, for regulatory function. We identified multiple accessible and functional regulatory DNA elements that harbor variants affecting their activity. Both variant regions drove gene expression in the cardiac conduction tissue in transgenic reporter mice. Genomic deletion from the mouse genome of one of the regions caused increased cardiac expression of only Tbx3, PR interval shortening and increased QRS duration. Combined, our findings address the mechanistic link between trait-associated variants in the gene desert, TBX3 regulation and cardiac conduction.

Targeted chromatin conformation analysis identifies novel distal neural enhancers of ZEB2 in pluripotent stem cell differentiation

The transcription factor zinc finger E-box binding protein 2 (ZEB2) controls embryonic and adult cell fate decisions and cellular maturation in many stem/progenitor cell types. Defects in these processes in specific cell types underlie several aspects of Mowat–Wilson syndrome (MOWS), which is caused by ZEB2 haplo-insufficiency. Human ZEB2, like mouse Zeb2, is located on chromosome 2 downstream of a ±3.5 Mb-long gene-desert, lacking any protein-coding gene. Using temporal targeted chromatin capture (T2C), we show major chromatin structural changes based on mapping in-cis proximities between the ZEB2 promoter and this gene desert during neural differentiation of human-induced pluripotent stem cells, including at early neuroprogenitor cell (NPC)/rosette state, where ZEB2 mRNA levels increase significantly. Combining T2C with histone-3 acetylation mapping, we identified three novel candidate enhancers about 500 kb upstream of the ZEB2 transcription start site. Functional luciferase-based assays in heterologous cells and NPCs reveal co-operation between these three enhancers. This study is the first to document in-cis Regulatory Elements located in ZEB2’s gene desert. The results further show the usability of T2C for future studies of ZEB2 REs in differentiation and maturation of multiple cell types and the molecular characterization of newly identified MOWS patients that lack mutations in ZEB2 protein-coding exons.