Genomic multidisciplinary teams: A model for navigating genetic mainstreaming and precision medicine.

AIM: Recent rapid advances in genomics are revolutionising patient diagnosis and management of genetic conditions. However, this has led to many challenges in service provision, education and upskilling requirements for non-genetics health-care professionals and remuneration for genomic testing. In Australia, Medicare funding with a Paediatric genomic testing item for patients with intellectual disability or syndromic features has attempted to address this latter issue. The Sydney Children's Hospitals Network - Westmead (SCHN-W) Clinical Genetics Department established Paediatric and Neurology genomic multidisciplinary team (MDT) meetings to address the Medicare-specified requirement for discussion with clinical genetics, and increasing genomic testing advice requests. METHODS: This SCHN-W genomic MDT was evaluated with two implementation science frameworks - the RE-AIM (Reach, Effectiveness, Adoption, Implementation, Maintenance) and GMIR - Genomic Medicine Integrative Research frameworks. Data from June 2020 to July 2022 were synthesised and evaluated, as well as process mapping of the MDT service. RESULTS: A total of 205 patients were discussed in 34 MDT meetings, facilitating 148 genomic tests, of which 73 were Medicare eligible. This was equivalent to 26% of SCHN-W genetics outpatient activity, and 13% of all Medicare-funded paediatric genomic testing in NSW. 39% of patients received a genetic diagnosis. CONCLUSION: The genomic MDT facilitated increased genomic testing at a tertiary paediatric centre and is an effective model for mainstreaming and facilitating precision medicine. However, significant implementation issues were identified including cost and sustainability, as well as the high level of resourcing that will be required to scale up this approach to other areas of medicine.

Honey bee neurogenomic responses to affiliative and agonistic social interactions.

Social interactions can be divided into two categories, affiliative and agonistic. How neurogenomic responses reflect these opposing valences is a central question in the biological embedding of experience. To address this question, we exposed honey bees to a queen larva, which evokes nursing, an affiliative alloparenting interaction, and measured the transcriptomic response of the mushroom body brain region at different times after exposure. Hundreds of genes were differentially expressed at distinct time points, revealing a dynamic temporal patterning of the response. Comparing these results to our previously published research on agonistic aggressive interactions, we found both shared and unique transcriptomic responses to each interaction. The commonly responding gene set was enriched for nuclear receptor signaling, the set specific to nursing was enriched for olfaction and neuron differentiation, and the set enriched for aggression was enriched for cytoskeleton, metabolism, and chromosome organization. Whole brain histone profiling after the affiliative interaction revealed few changes in chromatin accessibility, suggesting that the transcriptomic changes derive from already accessible areas of the genome. Although only one stimulus of each type was studied, we suggest that elements of the observed transcriptomic responses reflect molecular encoding of stimulus valence, thus priming individuals for future encounters. This hypothesis is supported by behavioral analyses showing that bees responding to either the affiliative or agonistic stimulus exhibited a higher probability of repeating the same behavior but a lower probability of performing the opposite behavior. These findings add to our understanding of the biological embedding at the molecular level.

Creating a mouse model resistant to induced ischemic stroke and cardiovascular damage.

Vascular prostanoids, isomerized from an intermediate prostaglandin (PG), H2, produced by cyclooxygenase (COX), exert various effects on the vascular system, both protective and destructive. During endothelial dysfunction, vascular protector prostacyclin/prostaglandin I2 (PGI2) is decreased, while inflammatory PGE2 and thrombotic TXA2 are increased. Therefore, our research aim was to reverse the event by controlling PGH2 metabolism by generating an in vivo model via enzymatic engineering of COX-1 and prostacyclin synthase (PGIS). The COX-1 and PGIS genes were linked to a 10-residue amino acid linker to form a Single-chain Enzyme Complex (SCHEC), COX-1-10aa-PGIS. Transgenic (CP-Tg) mice in a FVB/N background were generated using the pronuclear microinjection method. We first confirmed mRNA and protein expression of COX-1-10aa-PGIS in various CP-Tg mouse tissues, as well as upregulation of circulating PGI2. We then examined the cardiovascular function of these mice. Our CP-Tg mice exhibited marked resistance to vascular assault through induced carotid arterial blockage, acute thrombotic stroke and arterial arrest, angiotensin-induced peripheral vasoconstriction, and hepatic lipid accumulation after receiving a high-fat diet. They also had a longer lifespan compared with wild-type mice. This study raises the possibility of fighting cardiovascular diseases by regulating cellular arachidonic acid-derived PGH2 metabolites using enzymatic engineering.

The key residue within the second extracellular loop of human EP3 involved in selectively turning down PGE2- and retaining PGE1-mediated signaling in live cells.

Key residues and binding mechanisms of PGE1 and PGE2 on prostanoid receptors are poorly understood due to the lack of X-ray structures for the receptors. We constructed a human EP3 (hEP3) model through integrative homology modeling using the X-ray structure of the ß2-adrenergic receptor transmembrane domain and NMR structures of the thromboxane A2 receptor extracellular loops. PGE1 and PGE2 docking into the hEP3 model showed differing configurations within the extracellular ligand recognition site. While PGE2 could form possible binding contact with S211, PGE1 is unable to form similar contacts. Therefore, S211 could be the critical residue for PGE2 recognition, but is not a significant for PGE1. This prediction was confirmed using HEK293 cells transfected with hEP3 S211L cDNA. The S211L cells lost PGE2 binding and signaling. Interestingly, the S211L cells retained PGE1-mediated signaling. It indicates that S211 within the second extracellular loop is a key residue involved in turning down PGE2 signaling. Our study provided information that S211L within EP3 is the key residue to distinguish PGE1 and PGE2 binding to mediate diverse biological functions at the initial recognition step. The S211L mutant could be used as a model for studying the binding mechanism and signaling pathway specifically mediated by PGE1.

A survey of ancient conserved non-coding elements in the PAX6 locus reveals a landscape of interdigitated cis-regulatory archipelagos.

Biological differences between cell types and developmental processes are characterised by differences in gene expression profiles. Gene-distal enhancers are key components of the regulatory networks that specify the tissue-specific expression patterns driving embryonic development and cell fate decisions, and variations in their sequences are a major contributor to genetic disease and disease susceptibility. Despite advances in the methods for discovery of putative cis-regulatory sequences, characterisation of their spatio-temporal enhancer activities in a mammalian model system remains a major bottle-neck. We employed a strategy that combines gnathostome sequence conservation with transgenic mouse and zebrafish reporter assays to survey the genomic locus of the developmental control gene PAX6 for the presence of novel cis-regulatory elements. Sequence comparison between human and the cartilaginous elephant shark (Callorhinchus milii) revealed several ancient gnathostome conserved non-coding elements (agCNEs) dispersed widely throughout the PAX6 locus, extending the range of the known PAX6 cis-regulatory landscape to contain the full upstream PAX6-RCN1 intergenic region. Our data indicates that ancient conserved regulatory sequences can be tested effectively in transgenic zebrafish even when not conserved in zebrafish themselves. The strategy also allows efficient dissection of compound regulatory regions previously assessed in transgenic mice. Remarkable overlap in expression patterns driven by sets of agCNEs indicates that PAX6 resides in a landscape of multiple tissue-specific regulatory archipelagos.

Sequencing of Pax6 loci from the elephant shark reveals a family of Pax6 genes in vertebrate genomes, forged by ancient duplications and divergences.

Pax6 is a developmental control gene essential for eye development throughout the animal kingdom. In addition, Pax6 plays key roles in other parts of the CNS, olfactory system, and pancreas. In mammals a single Pax6 gene encoding multiple isoforms delivers these pleiotropic functions. Here we provide evidence that the genomes of many other vertebrate species contain multiple Pax6 loci. We sequenced Pax6-containing BACs from the cartilaginous elephant shark (Callorhinchus milii) and found two distinct Pax6 loci. Pax6.1 is highly similar to mammalian Pax6, while Pax6.2 encodes a paired-less Pax6. Using synteny relationships, we identify homologs of this novel paired-less Pax6.2 gene in lizard and in frog, as well as in zebrafish and in other teleosts. In zebrafish two full-length Pax6 duplicates were known previously, originating from the fish-specific genome duplication (FSGD) and expressed in divergent patterns due to paralog-specific loss of cis-elements. We show that teleosts other than zebrafish also maintain duplicate full-length Pax6 loci, but differences in gene and regulatory domain structure suggest that these Pax6 paralogs originate from a more ancient duplication event and are hence renamed as Pax6.3. Sequence comparisons between mammalian and elephant shark Pax6.1 loci highlight the presence of short- and long-range conserved noncoding elements (CNEs). Functional analysis demonstrates the ancient role of long-range enhancers for Pax6 transcription. We show that the paired-less Pax6.2 ortholog in zebrafish is expressed specifically in the developing retina. Transgenic analysis of elephant shark and zebrafish Pax6.2 CNEs with homology to the mouse NRE/Pα internal promoter revealed highly specific retinal expression. Finally, morpholino depletion of zebrafish Pax6.2 resulted in a "small eye" phenotype, supporting a role in retinal development. In summary, our study reveals that the pleiotropic functions of Pax6 in vertebrates are served by a divergent family of Pax6 genes, forged by ancient duplication events and by independent, lineage-specific gene losses.

Functional characterization of the human RPGR proximal promoter.

PURPOSE: Mutations in the retinitis pigmentosa (RP) GTPase regulator (RPGR) gene account for more than 70% of X-linked RP cases. This study aims to characterize the proximal promoter region of the human RPGR gene. METHODS: The 5'-flanking region (5 kb) of human RPGR was cloned and sequenced. A potential transcription start site and transcription factor binding motifs were identified by bioinformatic analysis. Constructs containing the putative human RPGR promoter region upstream of a luciferase reporter gene were generated and analyzed by transient transfection and luciferase assays. Transgenic mouse lines carrying a 3-kb human RPGR promoter sequence fused to lacZ were generated and RPGR proximal promoter activity was analyzed by X-gal staining. RESULTS: Bioinformatic analyses of the human RPGR 5'-flanking region uncovered potential transcription factor binding sites and a CpG island. Transient transfection assays with RPGR promoter/luciferase reporter constructs revealed a 980-bp fragment (-952 to +28) that produced higher levels of luciferase activity. Mutagenesis identified a putative Sp1 binding site that was critical for regulating transcriptional activity. We generated transgenic mice in which a lacZ reporter gene was controlled by the 3-kb upstream region of RPGR. ß-galactosidase expression was predominantly found in mouse retina, brain, and kidney. In the retina, the photoreceptor cell layer showed the strongest ß-galactosidase staining. CONCLUSIONS: Our study defined the human RPGR proximal promoter region in which a 3-kb fragment contained sufficient regulatory elements to control RPGR expression in mouse retina and other tissues. Characterization of the RPGR promoter will facilitate understanding of the functional role of RPGR in the retina and gene therapy of X-linked RP.