Building cross-border collaborations to increase diversity and accelerate rare disease drug development - meeting report from the inaugural IndoUSrare Annual Conference 2021.

The inaugural IndoUSrare Annual Conference was held virtually from 29 November to 2 December 2021 and was organized by the Indo US Organization for Rare Diseases (IndoUSrare). The event saw participation from over 250 stakeholders of rare diseases who joined in virtually by audio/video on the Zoom platform from around the world, with a majority of attendees concentrated in the Indian subcontinent and the United States. The conference was held over 4 days from 10:00 a.m. to 12:30 p.m. Eastern Time on each day, which accommodated participation by speakers and attendees from both the eastern and western hemispheres. The agenda over 4 days holistically covered broad topics of interest to different stakeholder groups such as representatives from organizations working toward policy frameworks for rare diseases or orphan drugs (Days 1, 4), biomedical research institutions (Day 2), patient advocacy organizations (Day 3), and patient advocacy and engagement offices within Industry (Day 4). In this meeting report, we summarize the key highlights from each day of this conference, with a perspective on future directions encouraging cross-border multistakeholder collaborations to maximize diversity, equity, and inclusion (DEI) in rare disease diagnosis, research, clinical trials, and treatment access. Each day included a keynote lecture on the theme of the day followed by a series of individual speaker presentations and/or a panel discussion. The goal was to understand current barriers and bottlenecks in the rare disease ecosystem. The discussions also helped highlight gaps and identify potential solutions that can be achieved through building multistakeholder collaborations across international borders, which we believe IndoUSrare is uniquely positioned to do with organizational programs such as rare patient foundation alliance, technology-enabled patient concierge, research corps, and corporate alliance program. The inaugural conference of the then 2+-year-old IndoUSrare organization laid the foundation for ongoing engagement of stakeholders between the two countries - the United States and India. The long-term goal is to scale the conference more broadly and serve as a model for other low- and middle-income countries (LMICs). Plain language summary: IndoUSrare held its inaugural Annual Conference from 29 November to 2 December 2021. It was focused on the theme of cross-border collaborations for rare disease drug development, with each day dedicated to a specific patient-focused discussion topic, ranging from patient-led advocacy (Advocacy Day), research (Research Day), rare disease community support and engagement (Patients Alliance Day), to industry collaborations (Industry Day). The 4-day conference was held in virtual mode and attracted over 250 attendees from across the globe. This meeting report provides the key highlights of the event and summarizes learnings and future directions encouraging cross-border collaborations to increase diversity, equity, and inclusion (DEI) in rare disease research and clinical trials.

Recent Advances in Systems and Network Medicine: Meeting Report from the First International Conference in Systems and Network Medicine.

The First International Conference in Systems and Network Medicine gathered together 200 global thought leaders, scientists, clinicians, academicians, industry and government experts, medical and graduate students, postdoctoral scholars and policymakers. Held at Georgetown University Conference Center in Washington D.C. on September 11-13, 2019, the event featured a day of pre-conference lectures and hands-on bioinformatic computational workshops followed by two days of deep and diverse scientific talks, panel discussions with eminent thought leaders, and scientific poster presentations. Topics ranged from: Systems and Network Medicine in Clinical Practice; the role of -omics technologies in Health Care; the role of Education and Ethics in Clinical Practice, Systems Thinking, and Rare Diseases; and the role of Artificial Intelligence in Medicine. The conference served as a unique nexus for interdisciplinary discovery and dialogue and fostered formation of new insights and possibilities for health care systems advances.

Differential gene expression and alternative splicing between diploid and tetraploid watermelon.

The exploitation of synthetic polyploids for producing seedless fruits is well known in watermelon. Tetraploid progenitors of triploid watermelon plants, compared with their diploid counterparts, exhibit wide phenotypic differences. Although many factors modulate alternative splicing (AS) in plants, the effects of autopolyploidization on AS are still unknown. In this study, we used tissues of leaf, stem, and fruit of diploid and tetraploid sweet watermelon to understand changes in gene expression and the occurrence of AS. RNA-sequencing analysis was performed along with reverse transcription quantitative PCR and rapid amplification of cDNA ends (RACE)-PCR to demonstrate changes in expression and splicing. All vegetative tissues except fruit showed an increased level of AS in the tetraploid watermelon throughout the growth period. The ploidy levels of diploids and the tetraploid were confirmed using a ploidy analyser. We identified 5362 and 1288 genes that were up- and downregulated, respectively, in tetraploid as compared with diploid plants. We further confirmed that 22 genes underwent AS events across tissues, indicating possibilities of generating different protein isoforms with altered functions of important transcription factors and transporters. Arginine biosynthesis, chlorophyllide synthesis, GDP mannose biosynthesis, trehalose biosynthesis, and starch and sucrose degradation pathways were upregulated in autotetraploids. Phloem protein 2, chloroplastic PGR5-like protein, zinc-finger protein, fructokinase-like 2, MYB transcription factor, and nodulin MtN21 showed AS in fruit tissues. These results should help in developing high-quality seedless watermelon and provide additional transcriptomic information related to other cucurbits.

Rare and common variants in extracellular matrix gene Fibrillin 2 (FBN2) are associated with macular degeneration.

Neurodegenerative diseases affecting the macula constitute a major cause of incurable vision loss and exhibit considerable clinical and genetic heterogeneity, from early-onset monogenic disease to multifactorial late-onset age-related macular degeneration (AMD). As part of our continued efforts to define genetic causes of macular degeneration, we performed whole exome sequencing in four individuals of a two-generation family with autosomal dominant maculopathy and identified a rare variant p.Glu1144Lys in Fibrillin 2 (FBN2), a glycoprotein of the elastin-rich extracellular matrix (ECM). Sanger sequencing validated the segregation of this variant in the complete pedigree, including two additional affected and one unaffected individual. Sequencing of 192 maculopathy patients revealed additional rare variants, predicted to disrupt FBN2 function. We then undertook additional studies to explore the relationship of FBN2 to macular disease. We show that FBN2 localizes to Bruch's membrane and its expression appears to be reduced in aging and AMD eyes, prompting us to examine its relationship with AMD. We detect suggestive association of a common FBN2 non-synonymous variant, rs154001 (p.Val965Ile) with AMD in 10 337 cases and 11 174 controls (OR = 1.10; P-value = 3.79 × 10(-5)). Thus, it appears that rare and common variants in a single gene--FBN2--can contribute to Mendelian and complex forms of macular degeneration. Our studies provide genetic evidence for a key role of elastin microfibers and Bruch's membrane in maintaining blood-retina homeostasis and establish the importance of studying orphan diseases for understanding more common clinical phenotypes.

Developing rods transplanted into the degenerating retina of Crx-knockout mice exhibit neural activity similar to native photoreceptors.

Replacement of dysfunctional or dying photoreceptors offers a promising approach for retinal neurodegenerative diseases, including age-related macular degeneration and retinitis pigmentosa. Several studies have demonstrated the integration and differentiation of developing rod photoreceptors when transplanted in wild-type or degenerating retina; however, the physiology and function of the donor cells are not adequately defined. Here, we describe the physiological properties of developing rod photoreceptors that are tagged with green fluorescent protein (GFP) driven by the promoter of rod differentiation factor, Nrl. GFP-tagged developing rods show Ca(2 +) responses and rectifier outward currents that are smaller than those observed in fully developed photoreceptors, suggesting their immature developmental state. These immature rods also exhibit hyperpolarization-activated current (Ih ) induced by the activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. When transplanted into the subretinal space of wild-type or retinal degeneration mice, GFP-tagged developing rods can integrate into the photoreceptor outer nuclear layer in wild-type mouse retina and exhibit Ca(2 +) responses and membrane current comparable to native rod photoreceptors. A proportion of grafted rods develop rhodopsin-positive outer segment-like structures within 2 weeks after transplantation into the retina of Crx-knockout mice and produce rectifier outward current and Ih upon membrane depolarization and hyperpolarization. GFP-positive rods derived from induced pluripotent stem (iPS) cells also display similar membrane current Ih as native developing rod photoreceptors, express rod-specific phototransduction genes, and HCN-1 channels. We conclude that Nrl-promoter-driven GFP-tagged donor photoreceptors exhibit physiological characteristics of rods and that iPS cell-derived rods in vitro may provide a renewable source for cell-replacement therapy.

Non-random mtDNA segregation patterns indicate a metastable heteroplasmic segregation unit in m.3243A>G cybrid cells.

Many pathogenic mitochondrial DNA mutations are heteroplasmic, with a mixture of mutated and wild-type mtDNA present within individual cells. The severity and extent of the clinical phenotype is largely due to the distribution of mutated molecules between cells in different tissues, but mechanisms underpinning segregation are not fully understood. To facilitate mtDNA segregation studies we developed assays that measure m.3243A>G point mutation loads directly in hundreds of individual cells to determine the mechanisms of segregation over time. In the first study of this size, we observed a number of discrete shifts in cellular heteroplasmy between periods of stable heteroplasmy. The observed patterns could not be parsimoniously explained by random mitotic drift of individual mtDNAs. Instead, a genetically metastable, heteroplasmic mtDNA segregation unit provides the likely explanation, where stable heteroplasmy is maintained through the faithful replication of segregating units with a fixed wild-type/m.3243A>G mutant ratio, and shifts occur through the temporary disruption and re-organization of the segregation units. While the nature of the physical equivalent of the segregation unit remains uncertain, the factors regulating its organization are of major importance for the pathogenesis of mtDNA diseases.

Transcriptome analysis using next generation sequencing reveals molecular signatures of diabetic retinopathy and efficacy of candidate drugs.

PURPOSE: To define gene expression changes associated with diabetic retinopathy in a mouse model using next generation sequencing, and to utilize transcriptome signatures to assess molecular pathways by which pharmacological agents inhibit diabetic retinopathy. METHODS: We applied a high throughput RNA sequencing (RNA-seq) strategy using Illumina GAIIx to characterize the entire retinal transcriptome from nondiabetic and from streptozotocin-treated mice 32 weeks after induction of diabetes. Some of the diabetic mice were treated with inhibitors of receptor for advanced glycation endproducts (RAGE) and p38 mitogen activated protein (MAP) kinase, which have previously been shown to inhibit diabetic retinopathy in rodent models. The transcripts and alternatively spliced variants were determined in all experimental groups. RESULTS: Next generation sequencing-based RNA-seq profiles provided comprehensive signatures of transcripts that are altered in early stages of diabetic retinopathy. These transcripts encoded proteins involved in distinct yet physiologically relevant disease-associated pathways such as inflammation, microvasculature formation, apoptosis, glucose metabolism, Wnt signaling, xenobiotic metabolism, and photoreceptor biology. Significant upregulation of crystallin transcripts was observed in diabetic animals, and the diabetes-induced upregulation of these transcripts was inhibited in diabetic animals treated with inhibitors of either RAGE or p38 MAP kinase. These two therapies also showed dissimilar regulation of some subsets of transcripts that included alternatively spliced versions of arrestin, neutral sphingomyelinase activation associated factor (Nsmaf), SH3-domain GRB2-like interacting protein 1 (Sgip1), and axin. CONCLUSIONS: Diabetes alters many transcripts in the retina, and two therapies that inhibit the vascular pathology similarly inhibit a portion of these changes, pointing to possible molecular mechanisms for their beneficial effects. These therapies also changed the abundance of various alternatively spliced versions of signaling transcripts, suggesting a possible role of alternative splicing in disease etiology. Our studies clearly demonstrate RNA-seq as a comprehensive strategy for identifying disease-specific transcripts, and for determining comparative profiles of molecular changes mediated by candidate drugs.

Next-generation sequencing facilitates quantitative analysis of wild-type and Nrl(-/-) retinal transcriptomes.

PURPOSE: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived retinal transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT-PCR) methods and to evaluate protocols for optimal high-throughput data analysis. METHODS: Retinal mRNA profiles of 21-day-old wild-type (WT) and neural retina leucine zipper knockout (Nrl(-/-)) mice were generated by deep sequencing, in triplicate, using Illumina GAIIx. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows-Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. qRT-PCR validation was performed using TaqMan and SYBR Green assays. RESULTS: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) and identified 16,014 transcripts in the retinas of WT and Nrl(-/-) mice with BWA workflow and 34,115 transcripts with TopHat workflow. RNA-seq data confirmed stable expression of 25 known housekeeping genes, and 12 of these were validated with qRT-PCR. RNA-seq data had a linear relationship with qRT-PCR for more than four orders of magnitude and a goodness of fit (R(2)) of 0.8798. Approximately 10% of the transcripts showed differential expression between the WT and Nrl(-/-) retina, with a fold change ≥1.5 and p value <0.05. Altered expression of 25 genes was confirmed with qRT-PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to retinal function. Data analysis with BWA and TopHat workflows revealed a significant overlap yet provided complementary insights in transcriptome profiling. CONCLUSIONS: Our study represents the first detailed analysis of retinal transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.