Inherited retinal degenerations (IRDs), although uncommon, are the primary cause of blindness among adults of working age. These disorders are genetically complex, involving over 300 different loci, and establishing a molecular diagnosis is crucial for access to emerging therapies and clinical trials. Standard panel-based next-generation sequencing (pNGS) identifies causative variants in approximately 70–80% of cases, leaving a significant subset unresolved. This study examines patients with negative first-tier pNGS results, highlighting the role of detailed clinical reassessment and the application of targeted second-tier genetic testing. By excluding individuals without IRDs and applying appropriate follow-up genetic analyses, we were able to determine a molecular cause in 56% of previously undiagnosed families, increasing the overall diagnostic yield to 92% (388/423). Our findings indicate that while pNGS remains the most cost-efficient initial strategy for diverse IRD populations, further testing should be guided by refined clinical evaluation—such as multimodal imaging and electrophysiology—and genetic indicators, including the presence of single alleles in recessive conditions, to achieve accurate and cost-effective diagnoses.
