Dynamic dysregulation of transcriptomic networks in brainstem autonomic nuclei during hypertension development in the female spontaneously hypertensive rat.

Neurogenic hypertension stems from an imbalance in autonomic function that shifts the central cardiovascular control circuits toward a state of dysfunction. Using the female spontaneously hypertensive rat and the normotensive Wistar-Kyoto rat model, we compared the transcriptomic changes in three autonomic nuclei in the brainstem, nucleus of the solitary tract (NTS), caudal ventrolateral medulla, and rostral ventrolateral medulla (RVLM) in a time series at 8, 10, 12, 16, and 24 wk of age, spanning the prehypertensive stage through extended chronic hypertension. RNA-sequencing data were analyzed using an unbiased, dynamic pattern-based approach that uncovered dominant and several subtle differential gene regulatory signatures. Our results showed a persistent dysregulation across all three autonomic nuclei regardless of the stage of hypertension development as well as a cascade of transient dysregulation beginning in the RVLM at the prehypertensive stage that shifts toward the NTS at the hypertension onset. Genes that were persistently dysregulated were heavily enriched for immunological processes such as antigen processing and presentation, the adaptive immune response, and the complement system. Genes with transient dysregulation were also largely region-specific and were annotated for processes that influence neuronal excitability such as synaptic vesicle release, neurotransmitter transport, and an array of neuropeptides and ion channels. Our results demonstrate that neurogenic hypertension is characterized by brainstem region-specific transcriptomic changes that are highly dynamic with significant gene regulatory changes occurring at the hypertension onset as a key time window for dysregulation of homeostatic processes across the autonomic control circuits.NEW & NOTEWORTHY Hypertension is a major disease and is the primary risk factor for cardiovascular complications and stroke. The gene expression changes in the central nervous system circuits driving hypertension are understudied. Here, we show that coordinated and region-specific gene expression changes occur in the brainstem autonomic circuits over time during the development of a high blood pressure phenotype in a rat model of human essential hypertension.

Conditional expression of endorepellin in the tumor vasculature attenuates breast cancer growth, angiogenesis and hyaluronan deposition.

The tumor stroma of most solid malignancies is characterized by a pathological accumulation of pro-angiogenic and pro-tumorigenic hyaluronan driving tumorigenesis and metastatic potential. Of all three hyaluronan synthase isoforms, HAS2 is the primary enzyme that promotes the build-up of tumorigenic HA in breast cancer. Previously, we discovered that endorepellin, the angiostatic C-terminal fragment of perlecan, evokes a catabolic mechanism targeting endothelial HAS2 and hyaluronan via autophagic induction. To explore the translational implications of endorepellin in breast cancer, we created a double transgenic, inducible Tie2CreERT2;endorepellin(ER)Ki mouse line that expresses recombinant endorepellin specifically from the endothelium. We investigated the therapeutic effects of recombinant endorepellin overexpression in an orthotopic, syngeneic breast cancer allograft mouse model. First, adenoviral delivery of Cre evoking intratumor expression of endorepellin in ERKi mice suppressed breast cancer growth, peritumor hyaluronan and angiogenesis. Moreover, tamoxifen-induced expression of recombinant endorepellin specifically from the endothelium in Tie2CreERT2;ERKi mice markedly suppressed breast cancer allograft growth, hyaluronan deposition in the tumor proper and perivascular tissues, and tumor angiogenesis. These results provide insight into the tumor suppressing activity of endorepellin at the molecular level and implicate endorepellin as a promising cancer protein therapy that targets hyaluronan in the tumor microenvironment.

A single cell transcriptomics map of paracrine networks in the intrinsic cardiac nervous system.

We developed a spatially-tracked single neuron transcriptomics map of an intrinsic cardiac ganglion, the right atrial ganglionic plexus (RAGP) that is a critical mediator of sinoatrial node (SAN) activity. This 3D representation of RAGP used neuronal tracing to extensively map the spatial distribution of the subset of neurons that project to the SAN. RNA-seq of laser capture microdissected neurons revealed a distinct composition of RAGP neurons compared to the central nervous system and a surprising finding that cholinergic and catecholaminergic markers are coexpressed, suggesting multipotential phenotypes that can drive neuroplasticity within RAGP. High-throughput qPCR of hundreds of laser capture microdissected single neurons confirmed these findings and revealed a high dimensionality of neuromodulatory factors that contribute to dynamic control of the heart. Neuropeptide-receptor coexpression analysis revealed a combinatorial paracrine neuromodulatory network within RAGP informing follow-on studies on the vagal control of RAGP to regulate cardiac function in health and disease.

3D single cell scale anatomical map of sex-dependent variability of the rat intrinsic cardiac nervous system.

We developed and analyzed a single cell scale anatomical map of the rat intrinsic cardiac nervous system (ICNS) across four male and three female hearts. We find the ICNS has a reliable structural organizational plan across individuals that provide the foundation for further analyses of the ICNS in cardiac function and disease. The distribution of the ICNS was evaluated by 3D visualization and data-driven clustering. The pattern, distribution, and clustering of ICNS neurons across all male and female rat hearts is highly conserved, demonstrating a coherent organizational plan where distinct clusters of neurons are consistently localized. Female hearts had fewer neurons, lower packing density, and slightly reduced distribution, but with identical localization. We registered the anatomical data from each heart to a geometric scaffold, normalizing their 3D coordinates for standardization of common anatomical planes and providing a path where multiple experimental results and data types can be integrated and compared.

Input-output signal processing plasticity of vagal motor neurons in response to cardiac ischemic injury.

Vagal stimulation is emerging as the next frontier in bioelectronic medicine to modulate peripheral organ health and treat disease. The neuronal molecular phenotypes in the dorsal motor nucleus of the vagus (DMV) remain largely unexplored, limiting the potential for harnessing the DMV plasticity for therapeutic interventions. We developed a mesoscale single-cell transcriptomics data from hundreds of DMV neurons under homeostasis and following physiological perturbations. Our results revealed that homeostatic DMV neuronal states can be organized into distinguishable input-output signal processing units. Remote ischemic preconditioning induced a distinctive shift in the neuronal states toward diminishing the role of inhibitory inputs, with concomitant changes in regulatory microRNAs miR-218a and miR-495. Chronic cardiac ischemic injury resulted in a dramatic shift in DMV neuronal states suggestive of enhanced neurosecretory function. We propose a DMV molecular network mechanism that integrates combinatorial neurotransmitter inputs from multiple brain regions and humoral signals to modulate cardiac health.

Innervation and Neuronal Control of the Mammalian Sinoatrial Node a Comprehensive Atlas.

[Figure: see text].

A Comprehensive Integrated Anatomical and Molecular Atlas of Rat Intrinsic Cardiac Nervous System.

We have developed and integrated several technologies including whole-organ imaging and software development to support an initial precise 3D neuroanatomical mapping and molecular phenotyping of the intracardiac nervous system (ICN). While qualitative and gross anatomical descriptions of the anatomy of the ICN have each been pursued, we here bring forth a comprehensive atlas of the entire rat ICN at single-cell resolution. Our work precisely integrates anatomical and molecular data in the 3D digitally reconstructed whole heart with resolution at the micron scale. We now display the full extent and the position of neuronal clusters on the base and posterior left atrium of the rat heart, and the distribution of molecular phenotypes that are defined along the base-to-apex axis, which had not been previously described. The development of these approaches needed for this work has produced method pipelines that provide the means for mapping other organs.

Novel Y Chromosome Retrocopies in Canids Revealed through a Genome-Wide Association Study for Sex.

The lack of an annotated reference sequence for the canine Y chromosome has limited evolutionary studies, as well as our understanding of the role of Y-linked sequences in phenotypes with a sex bias. In genome-wide association studies (GWASs), we observed spurious associations with autosomal SNPs when sex was unbalanced in case-control cohorts and hypothesized that a subset of SNPs mapped to autosomes are in fact sex-linked. Using the Illumina 230K CanineHD array in a GWAS for sex, we identified SNPs that amplify in both sexes but possess significant allele frequency differences between males and females. We found 48 SNPs mapping to 14 regions of eight autosomes and the X chromosome that are Y-linked, appearing heterozygous in males and monomorphic in females. Within these 14 regions are eight genes: three autosomal and five X-linked. We investigated the autosomal genes (MITF, PPP2CB, and WNK1) and determined that the SNPs are diverged nucleotides in retrocopies that have transposed to the Y chromosome. MITFY and WNK1Y are expressed and appeared recently in the Canidae lineage, whereas PPP2CBY represents a much older insertion with no evidence of expression in the dog. This work reveals novel canid Y chromosome sequences and provides evidence for gene transposition to the Y from autosomes and the X.

Exome sequencing reveals independent SGCD deletions causing limb girdle muscular dystrophy in Boston terriers.

BACKGROUND: Limb-girdle muscular dystrophies (LGMDs) are a heterogeneous group of inherited autosomal myopathies that preferentially affect voluntary muscles of the shoulders and hips. LGMD has been clinically described in several breeds of dogs, but the responsible mutations are unknown. The clinical presentation in dogs is characterized by marked muscle weakness and atrophy in the shoulder and hips during puppyhood. METHODS: Following clinical evaluation, the identification of the dystrophic histological phenotype on muscle histology, and demonstration of the absence of sarcoglycan-sarcospan complex by immunostaining, whole exome sequencing was performed on five Boston terriers: one affected dog and its three family members and one unrelated affected dog. RESULTS: Within sarcoglycan-δ (SGCD), a two base pair deletion segregating with LGMD in the family was discovered, and a deletion encompassing exons 7 and 8 was found in the unrelated dog. Both mutations are predicted to cause an absence of SGCD protein, confirmed by immunohistochemistry. The mutations are private to each family. CONCLUSIONS: Here, we describe the first cases of canine LGMD characterized at the molecular level with the classification of LGMD2F.

Beyond the MHC: A canine model of dermatomyositis shows a complex pattern of genetic risk involving novel loci.

Juvenile dermatomyositis (JDM) is a chronic inflammatory myopathy and vasculopathy driven by genetic and environmental influences. Here, we investigated the genetic underpinnings of an analogous, spontaneous disease of dogs also termed dermatomyositis (DMS). As in JDM, we observed a significant association with a haplotype of the major histocompatibility complex (MHC) (DLA-DRB1*002:01/-DQA1*009:01/-DQB1*001:01), particularly in homozygosity (P-val = 0.0001). However, the high incidence of the haplotype among healthy dogs indicated that additional genetic risk factors are likely involved in disease progression. We conducted genome-wide association studies in two modern breeds having common ancestry and detected strong associations with novel loci on canine chromosomes 10 (P-val = 2.3X10-12) and 31 (P-val = 3.95X10-8). Through whole genome resequencing, we identified primary candidate polymorphisms in conserved regions of PAN2 (encoding p.Arg492Cys) and MAP3K7CL (c.383_392ACTCCACAAA>GACT) on chromosomes 10 and 31, respectively. Analyses of these polymorphisms and the MHC haplotypes revealed that nine of 27 genotypic combinations confer high or moderate probability of disease and explain 93% of cases studied. The pattern of disease risk across PAN2 and MAP3K7CL genotypes provided clear evidence for a significant epistatic foundation for this disease, a risk further impacted by MHC haplotypes. We also observed a genotype-phenotype correlation wherein an earlier age of onset is correlated with an increased number of risk alleles at PAN2 and MAP3K7CL. High frequencies of multiple genetic risk factors are unique to affected breeds and likely arose coincident with artificial selection for desirable phenotypes. Described herein is the first three-locus association with a complex canine disease and two novel loci that provide targets for exploration in JDM and related immunological dysfunction.

A CHRNE frameshift mutation causes congenital myasthenic syndrome in young Jack Russell Terriers.

Congenital myasthenic syndromes (CMSs) are a group of rare genetic disorders of the neuromuscular junction resulting in structural or functional causes of fatigable weakness that usually begins early in life. Mutations in pre-synaptic, synaptic and post-synaptic proteins have been demonstrated in human cases, with more than half involving aberrations in nicotinic acetylcholine receptor (AChR) subunits. CMS was first recognized in dogs in 1974 as an autosomal recessive trait in Jack Russell Terriers (JRTs). A deficiency of junctional AChRs was demonstrated. Here we characterize a CMS in 2 contemporary cases of JRT littermates with classic clinical and electromyographic findings, and immunochemical confirmation of an approximately 90% reduction in AChR protein content. Loci encoding the 5 AChR subunits were evaluated using microsatellite markers, and CHRNB1 and CHRNE were identified as candidate genes. Sequences of the splice sites and exons of both genes revealed a single base insertion in exon 7 of CHRNE that predicts a frameshift mutation and a premature stop codon. We further demonstrated this pathogenic mutation in CHRNE in archival tissues from unrelated JRTs studied 34 years ago.

Acceptability and Accuracy of Cervical Cancer Screening Using a Self-Collected Tampon for HPV Messenger-RNA Testing among HIV-Infected Women in South Africa.

BACKGROUND: HIV increases women's risk for high-risk human papillomavirus (hrHPV) infection and invasive cervical cancer. South Africa has a high HIV prevalence but low cervical cancer screening coverage. Self-collection of cervical specimens and hrHPV testing, including hrHPV messenger-RNA (mRNA) testing, are methods aimed at increasing screening rates. However, data are limited on the acceptability and accuracy of tampon-based self-collection for hrHPV mRNA testing in HIV-infected women. METHODS: We recruited 325 HIV-infected women seeking care at a government HIV clinic in Pretoria, South Africa. A clinician performed a pelvic examination and obtained an endocervical specimen. Study participants performed self-collection using a tampon. Both clinician- and self-collected specimens were tested for hrHPV mRNA. Acceptability of both collection methods was assessed, the prevalence of hrHPV mRNA in our study population was estimated, test positivity of the two collection methods were compared, and test agreement was assessed by calculating the κ-statistic, sensitivity, and specificity. RESULTS: Over 90% of women reported no difficulties self-collecting specimens and 82% were willing to perform the tampon-collection at home. Based on clinician-collection specimens, the prevalence of hrHPV mRNA in our study population was 36.7% (95% CI: 31.4%- 42.0%). There was no difference in test positivity between clinician-collection, 36.7%, and tampon-collection, 43.5% (p-value = 0.08). Using clinician-collection as the reference test, the sensitivity and specificity for hrHPV mRNA of tampon-collection were 77.4% (95% CI: 69.8-85.0%) and 77.8% (95% CI: 71.9-83.6%), respectively. CONCLUSIONS: Tampon-based self-collection is acceptable to women and has similar hrHPV mRNA positivity rates as clinician-collection, but has reduced sensitivity and specificity compared to clinician-collection. The hrHPV mRNA prevalence in our study population is high, but similar to other high-risk populations, and highlights the need for improved cervical cancer screening. Further research into the optimal use of tampon-based collection as a cervical cancer screening tool is warranted.

A COLQ missense mutation in Labrador Retrievers having congenital myasthenic syndrome.

Congenital myasthenic syndromes (CMSs) are heterogeneous neuromuscular disorders characterized by skeletal muscle weakness caused by disruption of signal transmission across the neuromuscular junction (NMJ). CMSs are rarely encountered in veterinary medicine, and causative mutations have only been identified in Old Danish Pointing Dogs and Brahman cattle to date. Herein, we characterize a novel CMS in 2 Labrador Retriever littermates with an early onset of marked generalized muscle weakness. Because the sire and dam share 2 recent common ancestors, CMS is likely the result of recessive alleles inherited identical by descent (IBD). Genome-wide SNP profiles generated from the Illumina HD array for 9 nuclear family members were used to determine genomic inheritance patterns in chromosomal regions encompassing 18 functional candidate genes. SNP haplotypes spanning 3 genes were consistent with autosomal recessive transmission, and microsatellite data showed that only the segment encompassing COLQ was inherited IBD. COLQ encodes the collagenous tail of acetylcholinesterase, the enzyme responsible for termination of signal transduction in the NMJ. Sequences from COLQ revealed a variant in exon 14 (c.1010T>C) that results in the substitution of a conserved amino acid (I337T) within the C-terminal domain. Both affected puppies were homozygous for this variant, and 16 relatives were heterozygous, while 288 unrelated Labrador Retrievers and 112 dogs of other breeds were wild-type. A recent study in which 2 human CMS patients were found to be homozygous for an identical COLQ mutation (c.1010T>C; I337T) provides further evidence that this mutation is pathogenic. This report describes the first COLQ mutation in canine CMS and demonstrates the utility of SNP profiles from nuclear family members for the identification of private mutations.

Alleles of the major histocompatibility complex play a role in the pathogenesis of pancreatic acinar atrophy in dogs.

Exocrine pancreatic insufficiency (EPI) is a disease wherein pancreatic acinar cells fail to synthesize and secrete sufficient amounts of digestive enzymes for normal digestion of food. EPI affects many dog breeds, with a dramatically higher prevalence in the German shepherd dog (GSD) population. In this breed and perhaps others, EPI most often results from degeneration of the acinar cells of the pancreas, a hereditary disorder termed pancreatic acinar atrophy (PAA). Evidence of lymphocytic infiltration indicates that PAA is an autoimmune disease, but the genetic etiology remains unclear. Data from global gene expression and single nucleotide polymorphism profiles in the GSD suggest the involvement of the major histocompatibility complex [MHC; dog leukocyte antigen (DLA)]. To determine if alleles of the MHC influence development of EPI, genotyping of polymorphic class I (DLA-88) and II loci (DLA-DRB1, DLA-DQA1, and DLA-DQB1) was carried out for 70 affected and 63 control GSDs, and four-locus haplotypes were determined. One haplotype containing a novel allele of DLA-88 is very highly associated with EPI (OR > 17; P = 0.000125), while two haplotypes were found to confer protection from EPI (P = 0.00087 and 0.0115). Described herein is the genotyping of MHC class I and II loci in a GSD cohort, establishment of four-locus haplotypes, and association of alleles/haplotypes with EPI.

Genome-wide association studies for multiple diseases of the German Shepherd Dog.

The German Shepherd Dog (GSD) is a popular working and companion breed for which over 50 hereditary diseases have been documented. Herein, SNP profiles for 197 GSDs were generated using the Affymetrix v2 canine SNP array for a genome-wide association study to identify loci associated with four diseases: pituitary dwarfism, degenerative myelopathy (DM), congenital megaesophagus (ME), and pancreatic acinar atrophy (PAA). A locus on Chr 9 is strongly associated with pituitary dwarfism and is proximal to a plausible candidate gene, LHX3. Results for DM confirm a major locus encompassing SOD1, in which an associated point mutation was previously identified, but do not suggest modifier loci. Several SNPs on Chr 12 are associated with ME and a 4.7 Mb haplotype block is present in affected dogs. Analysis of additional ME cases for a SNP within the haplotype provides further support for this association. Results for PAA indicate more complex genetic underpinnings. Several regions on multiple chromosomes reach genome-wide significance. However, no major locus is apparent and only two associated haplotype blocks, on Chrs 7 and 12 are observed. These data suggest that PAA may be governed by multiple loci with small effects, or it may be a heterogeneous disorder.

The function of dog models in developing gene therapy strategies for human health.

The domestic dog is of great benefit to humankind, not only through companionship and working activities cultivated through domestication and selective breeding, but also as a model for biomedical research. Many single-gene traits have been well-characterized at the genomic level, and recent advances in whole-genome association studies will allow for better understanding of complex, multigenic hereditary diseases. Additionally, the dog serves as an invaluable large animal model for assessment of novel therapeutic agents. Thus, the dog has filled a crucial step in the translation of basic research to new treatment regimens for various human diseases. Four well-characterized diseases in canine models are discussed as they relate to other animal model availability, novel therapeutic approach, and extrapolation to human gene therapy trials.