Ophthalmology
Abstract
Despite advances in sequencing technologies, a molecular diagnosis remains elusive in many Mendelian disease patients. Current short-read clinical sequencing approaches cannot provide chromosomal phase information or epigenetic information without further sample processing, which is not routinely done and can result in an incomplete molecular diagnosis in patients. The ability to provide phased genetic and epigenetic information from a single sequencing run would improve the diagnostic rate of Mendelian conditions. Here we describe Targeted Long-read Sequencing of Mendelian Disease genes (TaLon-SeqMD) using a real-time adaptive sequencing approach. Optimization of bioinformatic targeting enabled selective enrichment of multiple disease-causing regions of the human genome. Haplotype-resolved variant calling and simultaneous resolution of epigenetic base modification could be achieved in a single sequencing run. The TaLon-SeqMD approach was validated in a cohort of 18 subjects with previous genetic testing targeting 373 inherited retinal disease (IRD) genes, yielding the complete molecular diagnosis in each case. This approach was then applied in two IRD cases with inconclusive testing, which uncovered non-coding and structural variants that were difficult to characterize by standard short-read sequencing. Overall, these results demonstrate TaLon-SeqMD as an approach to provide rapid phased-variant calling to provide the molecular basis of Mendelian diseases.
Authors
Kenji Nakamichi, Jennifer Huey, Riccardo Sangermano, Emily M. Place, Kinga M. Bujakowska, Molly Marra, Lesley A. Everett, Paul Yang, Jennifer R. Chao, Russell N. Van Gelder, Debarshi Mustafi
Abstract
Post-transplantation, T helper 1 (Th1)-mediated immune rejection is the predominant cause of graft failure. Th1 cell sensitization occurs through complex and context-dependent interaction among antigen-presenting cell subsets, particularly CD11b+ dendritic cells (DC2) and CD103+ dendritic cells (DC1). This interaction necessitates further investigation in context of transplant immunity. We use a well-established pre-clinical models of corneal transplantation and identified distinct roles of migratory CD103+ DC1 in influencing the outcomes of the grafted tissue. In recipients with uninflamed corneal beds, migratory CD103+DC1 demonstrate a tolerogenic phenotype that modulate the immunogenic capacity of CD11b+DC2 primarily mediated by IL-10, suppressing alloreactive CD4+Th1 cells via the PD-L1/PD-1 pathway, and enhancing Treg-mediated tolerance via αvβ8 integrin-activated TGFβ1, thus facilitating graft survival. Conversely, in recipients with inflamed and vascularized corneal beds, IFN-γ produced by CD4+Th1 cells induces migratory CD103+DC1 to adopt an immunostimulatory phenotype, characterized by the downregulation of regulatory markers including αvβ8 integrin and IL-10 and the upregulation of IL-12 and costimulatory molecules CD80/86, resulting in graft failure. The adoptive transfer of ex-vivo induced tolerogenic CD103+DC1(iDC1) effectively inhibits Th1 polarization and preserves the tolerogenic phenotype of their physiological counterparts. Collectively, our findings underscore the essential role played by CD103+DC1 in modulating host alloimmune responses.
Authors
Tomas Blanco, Hayate Nakagawa, Aytan Musayeva, Mark Krauthammer, Rohan Bir Singh, Akitomo Narimatsu, Hongyan Ge, Sara I. Shoushtari, Reza Dana
Abstract
Patient-specific induced pluripotent stem cell–derived (iPSC-derived) cell lines allow for therapies to be tailored to individual patients, increasing therapeutic precision and efficiency. Bietti crystalline dystrophy (BCD) is a rare blinding disease estimated to affect about 67,000 individuals worldwide. Here, we used iPSC-derived retinal pigment epithelium (iRPE) cells from patients with BCD to evaluate adeno-associated virus–mediated (AAV-mediated) gene augmentation therapy strategies. We found that BCD iRPE cells were vulnerable to blue light–induced oxidative stress and that cellular phenotype can be quantified using 3 robust biomarkers: reactive oxygen species (ROS), 4-hydroxy 2-nonenal (4-HNE) levels, and cell death rate. Additionally, we demonstrated that AAV-mediated gene therapy can significantly reduce light-induced cell death in BCD iRPE cells. This is the first proof-of-concept study to our knowledge to show that AAV-CYP4V2 gene therapy can be used to treat light-induced RPE damage in BCD. Furthermore, we observed significant variability in cellular phenotypes among iRPE from patients with BCD of divergent mutations, which outlined genotype-phenotype correlations in BCD patient–specific cell disease models. Our results reveal that patient-specific iRPE cells retained personalized responses to AAV-mediated gene therapy. Therefore, this approach can advance BCD therapy and set a precedent for precision medicine in other diseases, emphasizing the necessity for personalization in healthcare to accommodate individual diversity.
Authors
Yao Li, Richard R. Yang, Yong-Shi Li, Chun-Wei Hsu, Laura A. Jenny, Yang Kong, Merry Z.C. Ruan, Janet R. Sparrow, Stephen H. Tsang
Abstract
Malattia Leventinese/Doyne honeycomb retinal dystrophy (ML/DHRD) is an age-related macular degeneration–like (AMD-like) retinal dystrophy caused by an autosomal dominant R345W mutation in the secreted glycoprotein, fibulin-3 (F3). To identify new small molecules that reduce F3 production in retinal pigmented epithelium (RPE) cells, we knocked-in a luminescent peptide tag (HiBiT) into the endogenous F3 locus that enabled simple, sensitive, and high-throughput detection of the protein. The GSK3 inhibitor, CHIR99021 (CHIR), significantly reduced F3 burden (expression, secretion, and intracellular levels) in immortalized RPE and non-RPE cells. Low-level, long-term CHIR treatment promoted remodeling of the RPE extracellular matrix, reducing sub-RPE deposit-associated proteins (e.g., amelotin, complement component 3, collagen IV, and fibronectin), while increasing RPE differentiation factors (e.g., tyrosinase, and pigment epithelium-derived factor). In vivo, treatment of 8-month-old R345W+/+ knockin mice with CHIR (25 mg/kg i.p., 1 mo) was well tolerated and significantly reduced R345W F3-associated AMD-like basal laminar deposit number and size, thereby preventing the main pathological feature in these mice. This is an important demonstration of small molecule–based prevention of AMD-like pathology in ML/DHRD mice and may herald a rejuvenation of interest in GSK3 inhibition for the treatment of retinal degenerative diseases, including potentially AMD itself.
Authors
Sophia M. DiCesare, Antonio J. Ortega, Gracen E. Collier, Steffi Daniel, Krista N. Thompson, Melissa K. McCoy, Bruce A. Posner, John D. Hulleman
Abstract
Autosomal dominant optic atrophy plus (ADOA+) is characterized by primary optic nerve atrophy accompanied by a spectrum of degenerative neurological symptoms. Despite ongoing research, no effective treatments are currently available for this condition. Our study provided evidence for the pathogenicity of an unreported c.1780T>C variant in the OPA1 gene through patient-derived skin fibroblasts and an engineered HEK293T cell line with OPA1 downregulation. We demonstrated that OPA1 insufficiency promoted mitochondrial fragmentation and increased DRP1 expression, disrupting mitochondrial dynamics. Consequently, this disruption enhanced mitophagy and caused mitochondrial dysfunction, contributing to the ADOA+ phenotype. Notably, the Drp1 inhibitor, mitochondrial division inhibitor-1 (Mdivi-1), effectively mitigated the adverse effects of OPA1 impairment. These effects included reduced Drp1 phosphorylation, decreased mitochondrial fragmentation, and balanced mitophagy. Thus, we propose that intervening in DRP1 with Mdivi-1 could correct mitochondrial abnormalities, offering a promising therapeutic approach for managing ADOA+.
Authors
Yan Lin, Dongdong Wang, Busu Li, Jiayin Wang, Ling Xu, Xiaohan Sun, Kunqian Ji, Chuanzhu Yan, Fuchen Liu, Yuying Zhao
Abstract
Inherited retinal dystrophies (IRDs) are progressive diseases leading to vision loss. Mutation in the eyes shut homolog (EYS) gene is one of the most frequent causes of IRD. However, the mechanism of photoreceptor cell degeneration by mutant EYS has not been fully elucidated. Here, we generated retinal organoids from induced pluripotent stem cells (iPSCs) derived from patients with EYS-associated retinal dystrophy (EYS-RD). In photoreceptor cells of RD organoids, both EYS and G protein–coupled receptor kinase 7 (GRK7), one of the proteins handling phototoxicity, were not in the outer segment, where they are physiologically present. Furthermore, photoreceptor cells in RD organoids were vulnerable to light stimuli, and especially to blue light. Mislocalization of GRK7, which was also observed in eys-knockout zebrafish, was reversed by delivering control EYS into photoreceptor cells of RD organoids. These findings suggest that avoiding phototoxicity would be a potential therapeutic approach for EYS-RD.
Authors
Yuki Otsuka, Keiko Imamura, Akio Oishi, Kazuhide Asakawa, Takayuki Kondo, Risako Nakai, Mika Suga, Ikuyo Inoue, Yukako Sagara, Kayoko Tsukita, Kaori Teranaka, Yu Nishimura, Akira Watanabe, Kazuhiro Umeyama, Nanako Okushima, Kohnosuke Mitani, Hiroshi Nagashima, Koichi Kawakami, Keiko Muguruma, Akitaka Tsujikawa, Haruhisa Inoue
Abstract
Recent studies have uncovered that non-coding sequence variants may relate to Axenfeld-Rieger syndrome (ARS), a rare developmental anomaly with genetic heterogeneity. However, how these genomic regions are functionally and structurally associated with ARS is still unclear. In this study, we performed genome-wide linkage analysis and whole-genome sequencing in a Chinese ARS family and identified a heterozygous deletion of about 570 kb (termed LOH-1) in the intergenic sequence between PITX2 and FAM241A. Knockout of LOH-1 homologous sequences caused ARS phenotypes in mice. RNA-seq and RT-qPCR revealed a significant reduction in Pitx2 gene expression in LOH-1–/– mice, while Foxc1 expression remained unchanged. ChIP-seq and bioinformatics analysis identified a potential enhancer region (LOH-E1) within LOH-1. Deletion of LOH-E1 led to a significant downregulation of the PITX2 gene. Mechanistically, we found a sequence (hg38 chr4:111,399,594-111,399,691) which is on LOH-E1 could regulate PITX2 by binding to RAD21, a critical component of the cohesin complex. Knockdown of RAD21 resulted in reduced PITX2 expression. Collectively, our findings indicate that a potential enhancer sequence which is within LOH-1 may regulate PITX2 expression remotely through cohesin-mediated loop domains, leading to ARS when absent. 2
Authors
Yizheng Jiang, Yu Peng, Qi Tian, Zhe Cheng, Bei Feng, Junping Hu, Lu Xia, Hui Guo, Kun Xia, Liang Zhou, Zhengmao Hu
Abstract
Inflammatory lymphangiogenesis is intimately linked to immune regulation and tissue homeostasis. However, current evidence has suggested that classic lymphatic vessels are physiologically absent in intraocular structures. Here, we show that neolymphatic vessels were induced in the iris after corneal alkali injury (CAI) in a VEGFR3-dependent manner. Cre-loxP–based lineage tracing revealed that these lymphatic endothelial cells (LECs) originate from existing Prox1+ lymphatic vessels. Notably, the ablation of iridial lymphangiogenesis via conditional deletion of VEGFR3 alleviated the ocular inflammatory response and pathological T cell infiltration. Our findings demonstrate that iridial neolymphatics actively participate in pathological immune responses following injury and suggest intraocular lymphangiogenesis as a valuable therapeutic target for the treatment of ocular inflammation.
Authors
Zheng Liu, Keli Liu, Shunhua Shi, Xun Chen, Xinyu Gu, Weifa Wang, Keli Mao, Rukeye Yibulayi, Wanwen Wu, Lei Zeng, Weibin Zhou, Xiaofeng Lin, Feng Zhang, Bingsheng Lou
Abstract
AMP-activated protein kinase (AMPK) plays a crucial role in maintaining ATP homeostasis in photoreceptor neurons. AMPK is a heterotrimeric protein consisting of alpha, beta, and gamma subunits. The independent functions of the two isoforms of the catalytic alpha subunit, PRKAA1 and PRKAA2, are uncharacterized in specialized neurons such as photoreceptors. Here we demonstrate in mice that rod photoreceptors lacking PRKAA2, but not PRKAA1, show altered levels of cGMP, GTP, and ATP, suggesting isoform-specific regulation of photoreceptor metabolism. Furthermore, PRKAA2 deficient mice display visual functional deficits on electroretinography and photoreceptor outer segment structural abnormalities on transmission electron microscopy consistent with neuronal dysfunction, but not neurodegeneration. Phosphoproteomics identified inosine monophosphate dehydrogenase (IMPDH) as a molecular driver of PRKAA2-specific photoreceptor dysfunction, and inhibition of IMPDH improved visual function in Prkaa2 rod photoreceptor knockout mice. These findings highlight a novel, therapeutically targetable PRKAA2 isoform-specific function of AMPK in regulating photoreceptor metabolism and function through a previously uncharacterized mechanism affecting IMPDH activity.
Authors
Tae Jun Lee, Yo Sasaki, Philip A. Ruzycki, Norimitsu Ban, Joseph B. Lin, Hung-Ting Wu, Andrea Santeford, Rajendra S. Apte
Abstract
Traumatic optic neuropathy (TON) is a devastating condition that can occur after blunt or penetrating trauma to the head, leading to visual impairment or blindness. Despite these debilitating effects, no clinically available therapeutic targets neuroprotection or promotes axon regeneration in this or any optic neuropathy. Limited data in large animal models is a major obstacle to advancing treatments toward clinical therapeutics. To address this issue, we refined a surgical model of TON in Yucatan minipigs. First, we validated the model by demonstrating visual impairment by flash visual-evoked potential and retinal ganglion cell degeneration and death. Next, we developed and optimized a delivery method and non-toxic dosing of intravitreal brain-derived neurotrophic factor (BDNF) and cyclic adenosine monophosphate (cAMP). Finally, we showed that intravitreal injection of BDNF and cAMP rescued visual function and protected against retinal ganglion cell death and optic nerve axon degeneration. Together these data in a pre-clinical large animal model advance our understanding of and ability to model TON and further identify and develop candidate clinical therapeutics.
Authors
Kathleen Heng, Brent K. Young, BaoXiang Li, Ashley D. Nies, Xin Xia, Runxia R. Wen, Roopa Dalal, Gregory T. Bramblett, Andrew W. Holt, Jeffery M. Cleland, Jason N. Harris, Albert Y. Wu, Jeffrey L. Goldberg
No posts were found with this tag.
