Rapid genetic sequencing for inherited retinal disease
When attempting to identify the sequence of the genes responsible for inherited retinal diseases (IRDs), time is of the essence. Whereas previously, this process could take months, Assistant Professor Debarshi Mustafi, MD, PhD, and his colleagues in the UW Department of Ophthalmology have developed a method for genetic sequencing for IRDs that takes just days.
Mustafi, a pediatric retina specialist at Seattle Children’s with a lab at the Karalis Johnson Retina Center at South Lake Union, is investigating the genetic basis of inherited retinal degeneration and the potential for therapeutic intervention to prevent the progression of blindness. In the pediatric population, IRDs constitute a significant cause of visual impairment and can be one of the first presenting features of a syndromic condition with extra-ocular consequences.
Using isolated blood samples from affected IRD patients and their families, the lab performs targeted genome sequencing to identify novel pathogenic variants of disease and reconstruct disease haplotypes, with implications for interpreting disease risks in IRDs.
The molecular basis of IRDs requires effectively characterizing mutations across over 300 genes. Mustafi said that currently used short-read panel sequencing leaves 30–40 percent of patients with a non-diagnostic result. This is because smaller commercial panels do not cover all regions of disease genes. Targeted long-read sequencing can focus the sequencing efforts of disease regions of interest to provide comprehensive information on genetic variants contributing to disease.
Mustafi and his colleagues are now targeting “every gene implicated in causing disease” related to IRDs, enabling sequencing as many as 373 genes in one sequencing run.
“This sequencing has the potential to enable rapid, phased mutation analysis in a single step in a matter of days or hours,” he said. “The sequencing is customizable. It takes a matter of minutes to add or subtract a gene. This way, we can selectively sequence what we want and disregard everything else.”
The rapid turnaround from sample extraction to diagnosis will enhance clinicians' ability to provide more targeted therapy in a timely manner.
Mustafi is also investigating the potential of this technique to rapidly identify pathogenic variants in retinoblastoma, for which rapid diagnosis is critical as it can affect treatment regimens to minimize morbidity and mortality.
“Not only are we able to provide a complete molecular diagnosis in a shorter time frame, but we can do so at nearly a quarter of the cost of commercial panels and using only the blood from the patient,” Mustafi said. “The goal is to determine if we can identify variants quickly and think about the next step, knowing their disease variant we can target.”
Rapid sequencing is currently used to diagnose eye diseases such as Usher Syndrome, Stargardt disease, and retinitis pigmentosa. But there is potential to expand this to other ocular diseases and even extra-ocular inherited conditions, Mustafi said.
“We want to make the targeting sequencing more efficient and increase our ability to find rare variants,” he said. “This should also work on any Mendelian disease outside of ophthalmology.”
Dr. Mustafi grew up in Chicago and earned his bachelor’s degree with Honors in Chemistry from the University of Chicago. He then enrolled at Case Western Reserve University as part of the NIH Medical Scientist Training Program to earn his MD and PhD.
Upon completing his internship, he completed his ophthalmology residency at the University of Southern California/LA County. Upon finishing residency, he was awarded the prestigious Heed Fellowship and completed his medical and surgical vitreoretinal fellowship at the UW before joining the faculty.
