Early Diagnosis of Rare Diseases with a Focus on Pulmonary Arterial Hypertension: A Narrative Review

Health outcomes for rare diseases can be greatly affected by timely diagnosis.This paper presents a narrative review of current literature on rare diseases, with a focuson Pulmonary Arterial Hypertension (PAH), to identify needs for early diagnosisinitiatives. The review assessed: what needs to be done, what is currently being done,and what are the approaches or change theories that underlie these initiatives.Literature from online key-word searches included academic articles pertaining todiagnostic methods and physician surveys, and reports from advocacy groups andhealth authorities. Findings centred on the needs for: physician awareness/education,public awareness/education, research needs, consolidation of disease information, andthe need for system-wide early diagnosis strategies. Recommendations highlightedsteps to promote awareness and education among physicians and the public, investigatetheories of behaviour change, and develop and diffuse evaluation criteria of earlydiagnosis initiatives

Non-Coding-Regulatory Regions Of Human Brain Genes Delineated By Bacterial Artificial Chromosome Knock-In Mice

Background The next big challenge in human genetics is understanding the 98% of the genome that comprises non-coding DNA. Hidden in this DNA are sequences critical for gene regulation, and new experimental strategies are needed to understand the functional role of gene-regulation sequences in health and disease. In this study, we build upon our HuGX (\u27high-throughput human genes on the X chromosome’) strategy to expand our understanding of human gene regulation in vivo. Results In all, ten human genes known to express in therapeutically important brain regions were chosen for study. For eight of these genes, human bacterial artificial chromosome clones were identified, retrofitted with a reporter, knocked single-copy into the Hprt locus in mouse embryonic stem cells, and mouse strains derived. Five of these human genes expressed in mouse, and all expressed in the adult brain region for which they were chosen. This defined the boundaries of the genomic DNA sufficient for brain expression, and refined our knowledge regarding the complexity of gene regulation. We also characterized for the first time the expression of human MAOA and NR2F2, two genes for which the mouse homologs have been extensively studied in the central nervous system (CNS), and AMOTL1 and NOV, for which roles in CNS have been unclear. Conclusions We have demonstrated the use of the HuGX strategy to functionally delineate non-coding-regulatory regions of therapeutically important human brain genes. Our results also show that a careful investigation, using publicly available resources and bioinformatics, can lead to accurate predictions of gene expression

rAAV-compatible MiniPromoters for restricted expression in the brain and eye

Abstract Background Small promoters that recapitulate endogenous gene expression patterns are important for basic, preclinical, and now clinical research. Recently, there has been a promising revival of gene therapy for diseases with unmet therapeutic needs. To date, most gene therapies have used viral-based ubiquitous promoters–however, promoters that restrict expression to target cells will minimize off-target side effects, broaden the palette of deliverable therapeutics, and thereby improve safety and efficacy. Here, we take steps towards filling the need for such promoters by developing a high-throughput pipeline that goes from genome-based bioinformatic design to rapid testing in vivo. Methods For much of this work, therapeutically interesting Pleiades MiniPromoters (MiniPs; ~4 kb human DNA regulatory elements), previously tested in knock-in mice, were “cut down” to ~2.5 kb and tested in recombinant adeno-associated virus (rAAV), the virus of choice for gene therapy of the central nervous system. To evaluate our methods, we generated 29 experimental rAAV2/9 viruses carrying 19 different MiniPs, which were injected intravenously into neonatal mice to allow broad unbiased distribution, and characterized in neural tissues by X-gal immunohistochemistry for icre, or immunofluorescent detection of GFP. Results The data showed that 16 of the 19 (84 %) MiniPs recapitulated the expression pattern of their design source. This included expression of: Ple67 in brain raphe nuclei; Ple155 in Purkinje cells of the cerebellum, and retinal bipolar ON cells; Ple261 in endothelial cells of brain blood vessels; and Ple264 in retinal Müller glia. Conclusions Overall, the methodology and MiniPs presented here represent important advances for basic and preclinical research, and may enable a paradigm shift in gene therapy

Modelling Human Regulatory Variation in Mouse: Finding the Function in Genome-Wide Association Studies and Whole-Genome Sequencing

An increasing body of literature from genome-wide association studies and human whole-genome sequencing highlights the identification of large numbers of candidate regulatory variants of potential therapeutic interest in numerous diseases. Our relatively poor understanding of the functions of non-coding genomic sequence, and the slow and laborious process of experimental validation of the functional significance of human regulatory variants, limits our ability to fully benefit from this information in our efforts to comprehend human disease. Humanized mouse models (HuMMs), in which human genes are introduced into the mouse, suggest an approach to this problem. In the past, HuMMs have been used successfully to study human disease variants; e.g., the complex genetic condition arising from Down syndrome, common monogenic disorders such as Huntington disease and β-thalassemia, and cancer susceptibility genes such as BRCA1. In this commentary, we highlight a novel method for high-throughput single-copy site-specific generation of HuMMs entitled High-throughput Human Genes on the X Chromosome (HuGX). This method can be applied to most human genes for which a bacterial artificial chromosome (BAC) construct can be derived and a mouse-null allele exists. This strategy comprises (1) the use of recombineering technology to create a human variant–harbouring BAC, (2) knock-in of this BAC into the mouse genome using Hprt docking technology, and (3) allele comparison by interspecies complementation. We demonstrate the throughput of the HuGX method by generating a series of seven different alleles for the human NR2E1 gene at Hprt. In future challenges, we consider the current limitations of experimental approaches and call for a concerted effort by the genetics community, for both human and mouse, to solve the challenge of the functional analysis of human regulatory variation

High-throughput generation of regulatory allele series.

<p>(A) Human BAC RP11-144P8 was retrofitted seven times to generate the different regulatory variants (column 1). The method of retrofitting (column 2), targeting (column 3), and variant screening (column 4) is presented for each variant. Also given are the number of ESC clones isolated after electroporation (column 5), the number of correctly targeted clones after PCR validation using assays an average of 6 kb, and a maximum of 11 kb, apart (column 6), and the resulting percentage of correctly targeted clones (column 7). (B) Species-specific reverse transcriptase PCR demonstrates transcription from the human BAC in germline animals from four of the strains generated by the high-throughput approach. One-step reverse transcription PCR reactions were performed using oligonucleotides specific for human <i>NR2E1</i>, mouse <i>Nr2e1</i>, and mouse <i>Gapdh</i>. The results show, as expected, expression of the human <i>NR2E1</i> gene in adult eye, forebrain, and midbrain, but not in adult lung, heart, and liver. Marker, 100-bp ladder; positive control (Ctl+), human RNA for human <i>NR2E1</i> assay and mouse RNA for mouse <i>Nr2e1</i> and <i>Gapdh</i> assays; negative control (Ctl−), human RNA for mouse <i>Nr2e1</i> and <i>Gapdh</i> assays and mouse RNA for human <i>NR2E1</i> assay.</p

The literature is increasing more slowly for humanized mouse models than for GWASs and HWGS or novel mouse models.

<p>Interrogation of the PubMed literature database (<a href="http://www.ncbi.nlm.nih.gov/pubmed" target="_blank">http://www.ncbi.nlm.nih.gov/pubmed</a>) reveals a faster growing body of literature related to GWASs and HWGS (white bars) or novel mouse models (grey bars) than to HuMMs (black bars). Interrogation of the database was done using the online search option from EndNote (<a href="http://www.endnote.com/" target="_blank">http://www.endnote.com/</a>). Individual numbers of entries for the search terms “genome wide association studies” and “human whole genome sequencing” were added together for the figure. Search terms for novel mouse models were “novel knockout mouse”, “novel knockin mouse”, and “novel knock-in mouse”. The entries for the search term “humanized mouse models” were not restricted to genetic mouse models but included xenograft mouse models as well. Search terms were interrogated in “all fields” per year.</p

Mixed methods study exploring parent engagement in child health research in British Columbia

Objectives The objective of this study was to explore parent perspectives of and interest in an interactive knowledge translation platform called Child-Sized KT that proposes to catalyse the collaboration of patients, families, practitioners and researchers in patient-oriented research at British Columbia Children’s Hospital (BCCH). Methods An explanatory sequential mixed methods design was used over 1 year. Over 500 parents across BC completed an online survey, including a subsample of 102 parents who had accessed care at BCCH within the past 2 years. The survey explored parent perspectives about the value of their engagement at all stages of the research process and their interest in and concerns with using an online platform. Following the online survey, two focus groups were held with parents in the Vancouver area to explore themes emerging from the survey. Results Parents expressed keen interest in engaging in research at BCCH. Parents perceived benefit from their input at all stages of the research process; however, they were most interested in helping to identify the problem, develop the research question and share the results. Although parents preferred online participation, they had concerns about protecting the privacy of their child’s information. Conclusions Parents see value in their involvement in all stages of child health research at BCCH. Their input suggests that Child-Sized KT, a hypothetical online platform, would facilitate meaningful stakeholder engagement in child health research, but should offer a customised experience and ensure the highest standard of data privacy and protection.Pediatrics, Department ofPopulation and Public Health (SPPH), School ofPsychiatry, Department ofMedicine, Faculty ofOther UBCReviewedFacultyResearche