The Mustafi Lab is investigating the genetic basis of inherited retinal degenerations (IRDs) and potential for therapeutic intervention to prevent progression of blindness. By using patient-derived blood, we can isolate genomic DNA to carry out short- and long-read sequencing for haplotype reconstruction and variant identification in patients with incomplete diagnosis of an IRD. The pathogenic contribution of disease-causing variants can then be tested using cell culture systems and patient-derived induced pluripotent stem cells.

Current projects include:

1. Haplotype reconstruction and IRD disease variant identification using targeted long-read sequencing

Targeted enrichment 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. This allows our lab to generate comprehensive sequence coverage of pre-selected IRD gene loci.

Targeted sequencing allows for adequate coverage depth 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.

2. Demonstration of cellular effects of disease-causing variants using patient-derived stem cell and retinal organoid technology

Stem cells and organoid technology allow discovery of disease-causing variants and the pathogenicity of those variants. They also serve as a surrogate for human retina biopsy of diseased tissue. Phenotypic and functional consequences of possible disease alleles can be elucidated using techniques such as immunohistochemistry (IHC), electron microscopy (EM) and electrophysiology.

3. Determining pathogenicity of disease-causing variants with CRISPR-Cas9 editing

The final step to validate the pathogenicity of identified novel disease-causing variants is to establish their sufficiency and necessity This is done in patient-derived retinal organoids. CRISPR-Cas9 correction is applied to disease-causing variants to deduce their pathogenicity in IRDs.

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 treatment response in the future. For more information visit:

5. Rapid identification of 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.