Aniridia is panocular, congenital, and progressive disorder with an occurrence of approximately 1 in 83,000 live births ^{1, 2} . Aniridia is best characterized by the lack of or hypoplasia of the iris (for which it is named), in addition to several other ocular abnormalities, which culminate in reduced visual acuity ³ . Due to the progressive nature of the disease, individuals usually develop multiple ocular abnormalities, such as keratopathy, corneal vascularization and opacification, glaucoma, anterior chamber fibrosis, and cataracts ^{4– 7} . Although aniridia is most well known for its ocular phenotypes, the condition has a number of other abnormalities, including sensory, neural, cognitive, and auditory processing abnormalities ^{8, 9} .

The development of aniridia in humans is linked to heterozygous loss-of-function mutations to the PAX6 gene, which encodes a highly conserved transcription factor critical for normal eye and neural development ¹ . The vast majority of aniridia cases (80%) are associated with mutations in PAX6 ^{2, 10} . Functional mutations in this gene can be either sporadic or familial, and causal mutations in aniridia encompass a large number of variants. The majority of these variants are single nucleotide polymorphisms, which lead to a premature termination codon, and are found across the PAX6 locus ^{10, 11} . PAX6 is expressed in the developing eye, brain, and spinal cord, and is required for aspects of anatomical and functional development of the central nervous system (CNS) and visual system ¹² . Within the CNS, PAX6 is involved in patterning, regionalization, and the formation of neural circuits ^{13– 16} . Previous studies of patients with aniridia using structural magnetic resonance imaging (MRI) have shown abnormalities in major fiber tracts and subcortical structures of the brain, including the anterior commissure ^{9, 17– 20} , posterior commissure ^{18, 20} , corpus callosum ^{9, 19, 20} , pineal gland ^{18, 20} , optic chiasm ²⁰ , and olfactory bulb ^{17, 18} . The most consistently reported abnormalities are found in the anterior commissure, pineal gland, and optic chiasm; abnormalities in the posterior commissure, corpus callosum, and olfactory bulb are found in fewer than 35% of patients examined. Additionally, studies have shown conflicting results regarding grey matter volume differences in aniridia, with reports of both increases and decreases in whole brain grey matter ^{21, 22} . Most recently, it has been shown that there is an accelerated age-related increase in cortical thinning of the inferior parietal lobe and prefrontal/premotor areas in both brain hemispheres in aniridia compared to healthy patients ²³ .

While several previous studies have investigated structural brain abnormalities in patients with aniridia, the variance in brain structures affected and extent of anatomical abnormalities is high. The variance observed in the published literature may be interpreted as a result of genetic differences in patient samples, either directly related to disease-causing mutations or modifier effects caused by genomic differences across subjects. The current study sought to investigate gross anatomical correlates of aniridia in a new population sample. Results from this study will serve as a comparison for previous studies, as well as contribute to what is known about the distribution of neuroanatomical phenotypes in the aniridia population as a whole.

Whole brain analysis was used to identify major structural abnormalities in aniridia patients. All neuroanatomical results are reported in Table 1, which includes subject demographics and mutations. We analysed five major brain structures that demonstrate clear anatomical abnormalities in aniridia patients. Our study showed that all 12 aniridia patients had a reduced anterior commissure when compared to their demographically matched healthy comparisons, as shown in Figure 1. The posterior commissure was reduced in 5/12 aniridia patients and normal in 7/12 of aniridia patients ( Figure 1). Of the five aniridia patients with reduced posterior commissures, one had a reduced commissure at the midline. The pineal gland was affected in all 12 aniridia patients: absent in two ( Figure 1), highly reduced in one, and reduced in nine. The corpus callosum was slightly thinned in 3/12 aniridia patients and normal in 9/12 aniridia patients ( Figure 2). The optic chiasm was reduced in 7/12 of patients ( Figure 2) and normal in 5/12 patients.

In an effort to consider normal structural variation in the healthy population, we also evaluated healthy comparisons for structural brain abnormalities. We saw a reduced anterior commissure in two of the healthy comparisons and a reduced posterior commissure in one healthy comparison. The pineal gland showed the most variance within the healthy comparison group with one subject with no visible pineal gland, three healthy comparisons with slightly reduced to reduced pineal glands, and eight healthy comparisons with normal pineal glands. All healthy comparisons had normal corpus callosums and optic chiasms. A full description of which structures showed abnormalities in both the aniridia and healthy comparison groups are reported in Table 1. These findings provide context for asserting disease-related changes in the aniridia population, in the current study as well as others.

The most commonly reported neuroanatomical abnormality in MRI studies of aniridia patients is the anterior commissure. Previous studies have reported changes in the anterior commissure with some cases described as reductions and others reported as complete absence of the structure ^{9, 17– 19} . Consistent with these reports, our study identified a reduction in the anterior commissure in all 12 aniridia patients, but none of the patients lacked the structure. The posterior commissure has also been evaluated in previous studies: one study reports that it is present in all subjects while the other study presented evidence that one patient had an absent structure while the others had normal posterior commissures ^{18, 20} . We found no individuals lacking the posterior commissure, and fewer than half of our aniridia patients seemed to have an abnormal structure. Interestingly though, it seems as if one patient exhibited a reduction of the posterior commissure at the midline with thickened bundles adjacent to the midline suggesting that commissure formation was incomplete. PAX6 has a known role in formation of the posterior commissure in rodents, so it is likely that this phenotype in humans is a direct consequence of PAX6 deficiency ¹⁵ . In agreement with previous findings, we also see abnormal or absent pineal glands in our entire patient population. This finding is consistent with sleep regulation deficits in persons with aniridia reported in other studies ²⁵ . The corpus callosum has also been a structure commonly evaluated in aniridia MRI studies, with many reporting reductions in corpus callosum thickness and severe agenesis ^{9, 19, 20} . However, we found very few patients present with a reduced or abnormal corpus callosum, and propose that the slight reduction we see in three of our patients falls within normal population variation. Unlike most previous studies, we examined the optic chiasm, and found a reduction in the structure in more than half of our patients. This reduction could be a developmental consequence of the disorder or a progressive phenotype associated with reduced levels of PAX6. Anatomical abnormality findings seem to be highly dependent on population sample, and a larger collective sample in the literature will help us get closer to understanding common disease traits and variation.

As described above, we observed a reduction in the anterior commissure in every patient we evaluated and a reduction of the posterior commissure in five patients, but no individuals completely lacked either structure. Our study utilized a high resolution 3T MRI for data acquisition, while most other studies used a 1.5T MRI. Signal to noise ratio from 3T MRIs are almost double that of 1.5T MRIs, which will lead to an improved image quality and resolution from the 3T magnet ²⁶ . We propose that the difference in observing a reduced versus absent anterior/posterior commissure may be due to a difference in scan resolution between images captured from a 3T versus 1.5T magnet. We see multiple patients in our group who have a severely reduced anterior commissure, and identifying this abnormality using a 1.5T magnet may be more difficult than when using a 3T. Additionally, the posterior commissure is smaller than the anterior commissure naturally, making it more difficult to distinguish between presence and absence in a scan. Lower scan resolution may not capture small structures such as these commissures, especially if they are reduced in size, leading to a false judgment of their absence.

A recent study has found an age component to cortical thickness in aniridia patients when compared to healthy individuals. The study found that in patients with aniridia there is an accelerated reduction in cortical thickness of the inferior parietal lobe and prefrontal/premotor areas in both brain hemispheres ²³ . Adding to this age component seen in the Yogarajah (2016) study, there are also population differences in brain anatomy, even within healthy groups, between younger subjects and older subjects ²⁷ . Additionally, as we show in our study, there are anatomical abnormalities even within healthy, unaffected participants. This makes it vitally important for careful selection of comparison subjects, and presents a caveat for interpreting differences we see in this and other clinical populations.

In addition to abnormal structural findings in patient populations with aniridia, multiple studies have assessed volumetric differences in grey and white matter in the brains of aniridia versus healthy comparisons. Similar to the findings in gross structural differences, much variation exists between reports. Some studies show an increase in grey matter volume in aniridia patients compared to healthy control groups, while others find both increases and decreases depending on brain region ^{21, 22} . Changes in white matter findings follow the same suit with some reports of reductions in white matter and others finding both reductions and increases ^{21, 22} . Even more interestingly, structures, such as the anterior commissure, posterior commissure, and pineal gland, show no deviation from healthy in these group-wise comparisons. This suggests that either the abnormalities seen in these structures are not as common among aniridia patients as previously thought, or that the characteristics of anatomical changes observed in aniridia patients have a high degree of variability within the population. Due to the consistency of abnormalities in structures like the anterior commissure in our study, as well as others, we predict the latter explains this discrepancy.

The current study investigated anatomical brain abnormalities correlated to aniridia in a new population sample in an effort to serve as a comparison to previous studies. Our aim was to contribute to what is known about the distribution of neuroanatomical phenotypes in the aniridia population as a whole. Although we found similar neuroanatomical abnormalities as previous studies, we find the severity is not as great as previously reported. The anterior commissure and pineal gland seem to be the structures most affected in the aniridia patients we examined, and we do see abnormalities in the posterior commissure, corpus callosum and the optic chiasm, albeit at lower frequency than previously reported. We believe the neuroanatomical abnormalities seen in aniridia populations have a high level of variability, and future studies should be aimed at collecting more patient MRI scans so that the breadth of abnormalities can be assessed.

MRI sessions were completed after written informed consent was obtained and MRI safety screening was completed. All activities were approved by the Institutional Review Board of the University of Georgia prior to subject recruitment and data collection. All individuals who participated in this study provided consent for their demographic, mutation information, and images to be published.

The data referenced by this article are under copyright with the following copyright statement: Copyright: � 2017 Grant MK et al.

Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

Dataset 1: Aniridia and healthy comparison MRI data: Structural MRI DICOM files for 12 aniridia and 12 healthy comparison individuals. Files are in DICOM format and labeled according to subject I.D. found in Table 1. These files can be opened using SPM software run in Matlab or OsiriX DICOM viewer (see Methods section). doi, 10.5256/f1000research.11063.d153799 ²⁸

Mutation information that has been presented here is also available through PAX6 Allelic Variant Database ( http://lsdb.hgu.mrc.ac.uk/home.php?select_db=PAX6).