BackgroundHaploinsufficiency of the transcription factor PAX6 is the main cause of congenital aniridia, a genetic disorder characterized by iris and foveal hypoplasia. 11p13 microdeletions alteringPAX6or its downstream regulatory region (DRR) are present in about 25% of patients; however, only a few complex rearrangements have been described to date. Here, we performed nanopore-based whole-genome sequencing to assess the presence of cryptic structural variants (SVs) on the only two unsolved “PAX6-negative” cases from a cohort of 110 patients with congenital aniridia after unsuccessfully short-read sequencing approaches.ResultsLong-read sequencing (LRS) unveiled balanced chromosomal rearrangements affecting thePAX6 locusat 11p13 in these two patients and allowed nucleotide-level breakpoint analysis. First, we identified a cryptic 4.9 Mb de novo inversion disrupting intron 7 ofPAX6,further verified by targeted polymerase chain reaction amplification and sequencing and FISH-based cytogenetic analysis.Furthermore, LRS was decisive in correctly mapping a t(6;11) balanced translocation cytogenetically detected in a second proband with congenital aniridia and considered non-causal 15 years ago. LRS resolved that the breakpoint on chromosome 11 was indeed located at 11p13, disrupting the DNase I hypersensitive site 2 enhancer within the DRR ofPAX6,161 Kb from the causal gene. Patient-derived RNA expression analysis demonstratedPAX6haploinsufficiency, thus supporting that the 11p13 breakpoint led to a positional effect by cleaving crucial enhancers forPAX6transactivation. LRS analysis was also critical for mapping the exact breakpoint on chromosome 6 to the highly repetitive centromeric region at 6p11.1.ConclusionsIn both cases, the LRS-based identified SVs have been deemed the hidden pathogenic cause of congenital aniridia. Our study underscores the limitations of traditional short-read sequencing in uncovering pathogenic SVs affecting low-complexity regions of the genome and the value of LRS in providing insight into hidden sources of variation in rare genetic diseases.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40246-023-00490-8.