Background
Classic aniridia is a rare, usually inherited eye disorder characterized by partial or near-complete absence of the iris. Although the name refers to the iris, the condition often affects multiple parts of the eye, including the cornea, lens, retina, and optic nerve. Many patients experience reduced vision, light sensitivity, nystagmus, cataracts, glaucoma, and other complications that can worsen over time.
In most classic cases, aniridia is linked to changes involving the PAX6 gene, a key developmental gene that helps guide formation of the eye and other tissues. A person can have an abnormal PAX6 protein, or the gene may be structurally altered so that it can no longer be expressed properly. Importantly, not all disease-causing changes are simple sequence variants. Some are structural variants, such as deletions, duplications, inversions, or translocations. These can be difficult to detect with standard testing, especially when the overall amount of DNA is unchanged.
This report describes a 16-year-old boy with classic aniridia whose earlier clinical genetic testing had not found a cause. More advanced methods, including optical genome mapping and long-read whole-genome sequencing, finally revealed a very small intrachromosomal rearrangement involving PAX6. The case illustrates how newer genomic tools can diagnose patients who remain unexplained after routine testing.
Why Standard Testing Can Miss the Cause
Clinical evaluation of aniridia often begins with sequencing of PAX6 and analysis of its nearby regulatory region. If no variant is found, copy-number testing and broader genome sequencing may follow. However, short-read whole-genome sequencing can still miss some structural changes because it uses relatively short DNA fragments, making it harder to resolve complex breakpoints or balanced rearrangements.
Balanced rearrangements are especially challenging. In a copy number-neutral event, no net gain or loss of DNA is present, so typical deletion- or duplication-focused methods may appear normal. Yet the biological effect can be substantial if the rearrangement separates a gene from its essential regulatory elements. For genes like PAX6, which depend on distant control sequences, this can silence expression even when the coding sequence itself is intact.
Patient and Prior Testing
The patient was a 16-year-old male with classic aniridia. He had already undergone extensive clinical testing, including sequencing and copy-number analysis of PAX6 exons and the downstream regulatory region. He also had short-read whole-genome sequencing, which did not identify a definitive explanation for his condition.
Because the clinical picture strongly suggested a PAX6-related disorder despite negative results, additional research-based genomic testing was pursued. High-quality DNA extracted from blood was analyzed using optical genome mapping and long-read whole-genome sequencing. These methods are particularly useful for finding structural variants and for defining their exact breakpoints.
What Optical Genome Mapping Showed
Optical genome mapping identified a 55-kilobase deletion on chromosome 11p13 that included all PAX6 exons and exon 12 of ELP4. The deleted segment was not simply lost; instead, it had been inserted into 11q21. This indicated an intrachromosomal translocation, meaning the DNA segment moved within the same chromosome rather than to a different chromosome.
This finding was important because it explained why routine tests had not identified a classic deletion. The rearrangement was small and copy number-neutral overall, so the genome still appeared balanced in terms of total DNA content. Nevertheless, the location of the PAX6 coding region had changed, and that change could disrupt normal gene regulation.
What Long-Read Whole-Genome Sequencing Added
Long-read whole-genome sequencing defined the breakpoints with much greater precision. It confirmed that the downstream regulatory region required for normal PAX6 expression remained at the original 11p13 site. In other words, the PAX6 coding sequence had been moved away from the control elements that normally switch it on in the developing eye.
This is the key mechanism in this case. The translocated copy of PAX6 at 11q21 is expected to be functionally silent because it no longer sits next to the regulatory sequence needed for proper expression. Even though the gene sequence itself was not destroyed, it was separated from the instructions that tell it when and where to work.
Clinical Meaning of a Copy Number-Neutral Rearrangement
This case highlights an important principle in medical genetics: a gene can be disrupted without being deleted. When a structural variant preserves the amount of DNA but changes its arrangement, the effect may be invisible to tests that focus only on copy number.
For PAX6, this is especially relevant because its expression depends on a complex regulatory landscape. The gene has downstream elements that are essential for normal eye development. If a rearrangement moves PAX6 away from these elements, the result can resemble a loss-of-function mutation. Clinically, the outcome may be classic aniridia even though standard sequencing, deletion testing, and short-read genome analysis are unrevealing.
Why This Case Matters
According to the report, the identified rearrangement may represent the smallest structural variant described to date that separates the PAX6 coding region from its downstream regulatory domain. If so, it shows how even a relatively small chromosomal change can have major developmental consequences.
The case also underscores the limitations of short-read genome sequencing for difficult structural events. Short-read methods are powerful and widely used, but they may not detect small balanced translocations or precisely map complex breakpoints. Optical genome mapping and long-read sequencing can bridge that gap by revealing the architecture of the genome in much greater detail.
For patients with a strong clinical diagnosis of aniridia but negative routine testing, these technologies can provide a definitive answer. That can improve genetic counseling, clarify recurrence risk, and support more tailored long-term ophthalmic care.
Implications for Diagnosis
When classic aniridia is suspected, PAX6 testing remains the first step. If no pathogenic variant is found, clinicians should consider whether the case might involve a structural rearrangement rather than a simple sequence change. This is particularly true when the phenotype is strongly suggestive of PAX6 dysfunction.
In practice, advanced testing may include:
– Optical genome mapping to identify structural variants across large genomic regions
– Long-read whole-genome sequencing to resolve breakpoints and orientation
– Targeted assays to confirm rearrangements when needed
– Careful review of PAX6 regulatory regions, not just the coding exons
These methods are not necessary for every patient, but they can be crucial in unresolved cases.
Broader Lessons for Eye Genetics
Aniridia is one example of a broader pattern in medical genetics: gene regulation matters as much as gene coding. Developmental disorders can arise not only from mutations that alter protein structure, but also from changes in genomic architecture that disrupt enhancer-promoter communication or separate genes from their control elements.
This is why advances in genome technology are so important. A patient may have a clearly genetic condition with a classic phenotype, yet standard tests may appear negative. In such cases, the issue is often not the absence of a genetic cause, but the limitations of the testing strategy.
Limitations and Context
This report describes a single patient, so it cannot estimate how often this specific rearrangement occurs. It does, however, add to evidence that some unexplained aniridia cases may be due to small, balanced structural variants around PAX6. Further studies will be needed to determine how often optical genome mapping and long-read sequencing change the diagnosis in similar patients.
It is also worth noting that research-grade genomic analysis is not yet universally available in all clinical settings. As these technologies become more accessible, they may increasingly help solve cases that remain unresolved after routine genetic workup.
Conclusion
This case of classic aniridia was ultimately explained by a tiny intrachromosomal translocation involving PAX6. The rearrangement was copy number-neutral, which likely allowed it to escape detection by short-read whole-genome sequencing and other standard methods. Optical genome mapping and long-read whole-genome sequencing together revealed that the PAX6 coding region had been separated from its downstream regulatory elements, leading to loss of normal gene expression.
The report highlights an important diagnostic lesson: in patients with strong clinical evidence of PAX6-related aniridia but negative first-line testing, advanced structural genome analysis may be necessary for a definitive diagnosis. It also demonstrates how noncoding regulatory architecture can be just as important as the gene itself in human disease.