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The Mustafi Lab is investigating the genetic basis of inherited retinal conditions and potential for therapeutic intervention to prevent progression of blindness. By using patient-derived samples (blood and saliva), we can isolate genomic DNA to carry out targeted long-read sequencing for haplotype reconstruction and variant identification in patients with incomplete diagnosis of inherited retinal diseases and retinoblastoma. The pathogenic contribution of disease-causing variants can then be tested using cell culture systems and patient-derived induced pluripotent stem cells to benchmark a pre-clinical therapeutic strategy for treating pediatric inherited diseases.
Current projects include:
1. TaLon-SeqMD: Targeted Long-read Sequencing of Mendelian Disease genes
Targeted enrichment using adaptive sampling avoids wasting sequencing bandwidth on uninformative reads to allow much deeper coverage from the same sequencing effort. By aligning raw Nanopore signal to an in silico representation of a reference sequence utilizing GPU base-calling, and resetting individual nanopores processing DNA outside the region of interest in real-time, on-the-fly target enrichment can be achieved.
Targeted sequencing allows for focused coverage of the disease gene of interest for haplotyping. Variants can be obtained from a haplotype-aware genotyping pipeline to narrow down potential pathogenic candidates without a priori knowledge of the disease-causing variants. This approach used in the lab can establish a diagnosis from the proband without the need for familial segregation.
Relevant Publications:
Nakamichi K, Huey J, Sangermano R, Place EM, Bujakowska KM, Marra M, Everett LA, Yang P, Chao JR, Van Gelder RN, Mustafi D. Targeted long-read sequencing enriches disease-relevant genomic regions of interest to provide complete Mendelian disease diagnostics. JCI Insight. 2024 Sep 12:e183902. doi: 10.1172/jci.insight.183902. PMID: 39264853. [Pubmed] [PDF]
Gupta P, Nakamichi K, Bonnell AC, Yanagihara R, Radulovich N, Hisaka F, Chao JR, Mustafi D. Familial co-segregation and the emerging role of long-read sequencing to re-classify variants of uncertain significance in inherited retinal diseases. NPJ Genomic Medicine, 2023, 8(20): doi: 10.1038/s41525-023-00366-9. PMID: 37558662. [Pubmed] [PDF]
Nakamichi K, Van Gelder RN, Chao JR, Mustafi D. Targeted adaptive long-read sequencing for discovery of complex phased variants in inherited retinal disease patients. Scientific Reports, 2023,3(1):8535. PMID: 37237007. [PubMed] [PDF]
2. Rapid identification of parent-of-origin disease variants in Retinoblastoma
In collaboration with Dr. Andrew Stacey at UW/SCH we are leveraging our ability to carry out targeted long-read sequencing of genomic DNA from patients with retinoblastoma (RB) to carry out haplotagging for the accurate identification of the chromosomal architecture of identified disease variants. This approach is minimally invasive and provides adequate depth of coverage to identify rare variants that contribute to disease. Moreover, targeted long-read sequencing offers a rapid and economical approach to variant discovery in RB.
Relevant Publications:
Stacey AW, Nakamichi K, Huey J, Stevens J, Waligorski N, Crotty EE, Van Gelder RN, Mustafi D. Prognostic importance of direct assignment of parent-of-origin via long-read genome and epigenome sequencing in retinoblastoma. JCI Insight. 2024 Dec 26:e188216. PMID: 39724000. [Pubmed] [PDF]
Nakamichi K, Stacey AW, Mustafi D. Targeted long-read sequencing allows for rapid identification of pathogenic disease-causing variants in retinoblastoma. Ophthalmic Genet, 2022, 43(6):762-770. PMID: 36325802. [PubMed] [PDF]
Mustafi D, Waligorski N, Cole B, Paulson V, Crotty EE, Stacey AW. Pineoblastoma without retinal tumors in a patient with a mosaic retinoblastoma pathogenic variant. JAMA Ophthalmology 2022, 140(4): 437-439, PMID: 35297960. [PubMed] [PDF]
3. Demonstration of cellular effects of disease-causing variants using patient-derived stem cell and retinal organoid technology
The goal of this project is to improve our genetic, mechanistic, and therapeutic understanding of IRDs using patient-derived retinal organoids. In collaboration with Dr. Timothy Cherry at SCRI we are looking to characterize variants of uncertain significance by modeling the unsolved IRDs with patient-derived retinal organoids. We expect that patient-specific organoid models will reveal the functional impact of VUSs and the molecular and cellular pathways affected in IRDs. Finally we plan to correct patient-variants using rapid CRISPR electroporation to determine the functional impact specific variants.
Relevant Publications:
Nakamichi K, Huey J, Sangermano R, Place EM, Bujakowska KM, Marra M, Everett LA, Yang P, Chao JR, Van Gelder RN, Mustafi D. Targeted long-read sequencing enriches disease-relevant genomic regions of interest to provide complete Mendelian disease diagnostics. JCI Insight. 2024 Sep 12:e183902. doi: 10.1172/jci.insight.183902. PMID: 39264853. [Pubmed] [PDF]
Mustafi D, Bharathan SP, Calderon R, Nagiel A. Human Cellular Models for Retinal Disease: From iPSCs to Organoids. Retina, 2022, 42(10):1829-1835. PMID: 35858274. [PubMed] [PDF]
4. Identifying the precise phenotypic and functional signatures of disease in IRDs using non-invasive adaptive optics based imaging
In collaboration with Dr. Ramkumar Sabesan's laboratory at UW, we are utilizing non-invasive adaptive optics based imaging of the retina to obtain more precise structural and functional features of photoreceptors during the degenerative process in various IRDs. A novel tool, termed optoretinography, is able to measure the optical signature of light-induced electrophysiological activity of photoreceptors to enable a single cell level readings of photoreceptor functionality. This tool can help understand the earliest features of retinal degeneration in IRDs and be an invaluable tool to track future treatment response.
Relevant Publications:
Wendel B, Pandiyan VP, Liu T, Jiang X, Lassoued A, Slezak S, Schleuffer S, Tutem WS, Mustafi D, Chao JR, Sabesan R. Multi-modal high-resolution imaging in retinitis pigmentosa: a comparison between optoretinography, cone density and visual sensitivity. IOVS. 2024 Aug 1;65(10):45. PMID: 39207297. [Pubmed] [PDF]
Huey J, Gupta P, Wendel B, Liu T, Bharadwaj P, Schwartz H, Kelly JP, Chang I, Chao JR, Sabesan R, Nagiel A, Mustafi D. Genetic Reasons for Phenotypic Diversity in Neuronal Ceroid Lipofuscinoses and High-resolution Imaging as a Marker of Retinal Disease. Ophthalmology Science. 2024 May 29;4(6):100560. [Pubmed] [PDF]
5. Multi-modal imaging to reveal disease signatures of pediatric retinal conditions
Our lab is utilizing multi-modal imaging approaches to better identify subtle disease signatures in pediatric retinal conditions. We have demonstrated that incorporation of oral fluorescein angiography into clinical practice can better allows one to track disease progression over time to direct disease-modifying therapies based on objective measures. By correlating biomarkers of disease with imaging characteristics, we are looking to provide clinicians with advanced methods to diagnose pediatric retinal conditions.
Relevant Publications:
Lindquist M, Cech J, Peterson A, Huey J, Mustafi D. Genetic counselor integration improves genetic eye disease diagnostics. Ophthalmology Retina, 2025 Mar 23:S2468-6530(25)00125-3. [Pubmed] [PDF]
Yanagihara R, Mustafi D, Huang LC. Cytomegalovirus retinitis response in an adolescent treated with systemic maribavir. Ophthalmology. 2024 Nov 4:S0161-6420(24)00603-1. PMID: 39503661. [Pubmed] [PDF]
Sawyer C, Huang LC, Vincent J, Cabrera MT, Herlihy E, Mustafi D. Diagnostic Role of Oral Fluorescein Angiography in Pediatric Ambulatory Clinics. Ophthalmology Retina, 2024 Feb;8(2):204-206. PMID: 39491131.[Pubmed] [PDF]
Mustafi D, Huang J, Ting MA, Waligorski N, Stacey AW, Huang LC. Successful treatment response of a juxtapapillary retinal capillary hemangioblastoma due to von Hippel-Lindau with Belzutifan in a pediatric patient. Retina, 2024 May 1;44(5):e31-e33. [Pubmed] [PDF]
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