Phenotypic and genotypic heterogeneity, especially in ASD, have created challenges for proper diagnosis and classification of disease [3]. Genes involving both autosomal recessive and autosomal dominant patterns of inheritance have been described with a wide phenotypic variability and expressivity.

Indeed, the introduction of NGS has revolutionized the field of human genetics, increasing the opportunity to establish molecular diagnoses and identify new associated genes. The usefulness of genetic testing by NGS is manifold: (i) it provides a more precise diagnosis, especially in the neonatal period when the phenotype may not yet have fully manifested; (ii) it can be used for carrier screening, which widens the choice of reproductive options for those who are diagnosed as carriers, (iii) it could foster the development of novel treatments that are genotype specific [1]. Notwithstanding, genetic testing comes with great challenges mostly related to variant interpretation.

In the last 20 years, we collected a cohort of 162 patients affected by ocular dysgenesis, mostly aniridia.

Our data certainly reiterate the notion that PAX6 alterations are primarily associated with ASD, since the majority of the cohort (66.7%) has a pathogenic or likely pathogenic variant in the PAX6 locus. This percentage gets an obvious increase by analyzing only isolated or syndromic aniridia cases (82.2%). Indeed, previous literature clearly demonstrated that PAX6 alterations explain about 80% of aniridia patients, both sporadic and familial [31]. The landscape of PAX6 coding variants is similar to that reported in literature [18, 32] even though we assessed a small augmentation in splice site variants (25.4% vs 15%). Exon 5 is proven to include the largest number of variants, a foreseeable event given that exons 5 and 6 encode for the paired domain, one of the PAX6 DNA-binding domains that has critical roles in development of the eye, the pancreas, and the central nervous system [33]. What proves to be worthy of attention is that our cohort included a striking fraction of deletions involving the 11p13 locus (20.8% of all PAX6-positive samples), either partially/totally involving PAX6 coding region or abolishing its critical regulatory region (Fig. 4, Table 1). PAX6 is surrounded by CREs that spatially and temporally direct its expression at different developmental stages. The large PAX6 regulatory landscape contains several enhancers that act in a finely controlled manner to direct PAX6 expression in the developing central nervous system, retina, lens, olfactory bulb, and pancreas [16, 17, 34].

Schematic representation of 11p13 deletions assessed in our cohort. 3′ of PAX6. Genes are represented by light gray and gray boxes. Arrows indicate the direction of transcription. Light green eclipses represent enhancers. Dark green eclipses represent SIMO and E180B. Black lines represent pathogenic and likely pathogenic deletions found in our cohort, while the orange line represents the VUS. Vertical dashed lines represent the 244 kb and the 18 kb PAX6 critical regulatory regions identified by [20] and [21], respectively. Some of these deletions are also described in [13]. Genomic coordinates are based on human genome assembly hg19

The regulatory role of these CREs was suggested by the existence of aniridia in patients with different chromosome 11p13 rearrangements affecting the downstream elements while preserving the PAX6 coding sequence [21, 35]. Therefore, over the past 15 years a significant part of the ophthalmologic research has focused on characterizing these CREs. Given the abundance of regulatory sequences surrounding PAX6, some amount of overlap among the regulatory sequences in directing PAX6 in specific tissues has always been assumed [16]. Indeed whether these enhancers perform additive, redundant, or distinct functions is largely unknown. Studies in zebrafish have allowed to recognize specific spatio-temporal activity patterns for some regulatory sequences [36, 37]. Uttley et al. demonstrated using dual enhancer–reporter zebrafish embryos that the two PAX6 retinal enhancers HS5 and NRE have both overlapping and spatio-temporal specific activities [37]. Besides, with an exception in the SIMO lens enhancer, deleterious point mutation affecting these CREs appears to be an ultra-rare event. All these assumptions emphasize how little we actually know about how these enhancers work in regulating PAX6 expression. Furthermore, which is the minimal regulatory region that if deleted is able to elicit aniridia is still debated. To date, a large number of 11p13 deletions have been already identified [21, 38, 39], but correlation with phenotypes is still scanty. In 2016, Ansari et al. postulated a 244 kb critical region required for PAX6 transcriptional activation [20]. From this time forward, several papers assessed smaller deletions, partially involving ELP4, in patients affected by aniridia [21, 40].

This turns out to be of particular interest by analyzing one of the VUS identified in this cohort, i.e., a 260.8 kb deletion assessed in a patient with familial aniridia (A114) involving part of the ELP4 gene (int2-3 to int9-10). The deletion removes at least 10 PAX6 CREs including RB, E180B, HS2-8, E100, E120, SIMO, and E60 [19, 21], but does not entirely erase the 244 kb critical region proposed by Ansari et al. [20]. Plaisanciè et al. proposed an 18 kb minimal region including the E180B enhancer that, if deleted, correlates with the aniridia phenotype without extraocular manifestations. Our deletion removes an interaction-rich region toward both 3′ and 5′, which includes both SIMO and E180B, so its involvement in the patient’s phenotype would not be surprising. However, these are assumptions that will have to be validated by future studies, perhaps in vivo.

The use of WES has allowed us to expand the analytical portfolio and identify rarer phenotypes. Indeed, multiple conditions and syndromes are categorized under the umbrella of ASD [41], and nowadays at least 60 genes have been associated with this condition [5]. SNVs in genes strongly associated with ocular dysgenesis have been found in our cohort, including ACTA2, CYP1B1, FZD5, PXDN, PITX2, and TFAP2A. Besides PAX6, the second most altered gene in our cohort turned out to be ITPR1, whose either homozygous or heterozygous, dominant-negative, pathogenic variants are associated to the Gillespie syndrome—characterized by a triad of partial aniridia, non-progressive cerebellar ataxia, and intellectual disability [42]. Gillespie syndrome is an exceptionally uncommon diagnosis with <50 patients ever being diagnosed [43]. None of our patients (A210, A253, and A258) presented the classical triad of symptoms, with two out of three patients affected by intellectual disability. No signs of ataxia were detected. This is not surprising given that papers describing patients with atypical presentations have been only recently published [43]. Furthermore, two MAB21L1 variants were found in our cohort, the hotspot mutation p.Arg51Leu and the previously published p.Phe52Cys (A180 and A97, respectively) [22]. Indeed, MAB21L1 is gaining momentum as a novel gene associated with severe aniridia and/or microphthalmia [22, 44].

Furthermore, WES allowed us to diagnose severe syndromic conditions such as the MIDAS syndrome, the oculogastrointestinal-neurodevelopmental syndrome and the JBS. MIDAS (microphthalmia, dermal aplasia, and sclerocornea) syndrome has been described 30 years ago. Ocular findings commonly include microphthalmia and sclerocornea even though corneal opacities without sclerocornea, microcornea, corneal leukoma, congenital glaucoma, aniridia, cataract, and Peters’ anomaly have been described. It is caused by Xp22.2 deletions and it is characterized by wide inter- and intra-familial phenotypic variability, which has been associated with skewed X inactivation of the genes involved [45]. Concerning the proband (A84), skin defects were not reported at birth. Some unusual MIDAS presentations could be assessed in literature, with eye abnormalities in the absence of skin defects and vice versa [23].

To date, oculogastrointestinal-neurodevelopmental syndrome has been described in less than ten published individuals, and it is associated with biallelic pathogenic variants in the CAPN15 gene [25]. While loss of function variants are associated to a more severe phenotype including ocular defects, microcephaly, craniofacial abnormalities, cardiac and genitourinary malformations, abnormal neurologic activity, and developmental delay, missense variants rise a milder phenotype mostly characterized by microphthalmia and/or coloboma in association with mild developmental delay [46]. All this is in accordance with our patient’s phenotype (A106).

Lastly, the JBS is a rare and poorly understood multisystem genomic disorder where the distal region of chromosome 11q is deleted. It is a quite rare condition characterized by multiple anomalies including developmental delay, craniofacial dysmorphisms, craniosynostosis, ocular abnormalities, congenital heart disease, intellectual disability, Paris Trousseau hemorrhagic disease, and immunodeficiency [47]. Notwithstanding, clinical manifestations depend on the size of deletion, which usually varies between 7 and 20 Mb [48]. Our patient (A237) bore a 1.2 Mb deletion involving only the 11q25 cytoband, which probably does not result in a full-blown JBS phenotype. Indeed, the 11q24 locus has been associated with the thrombocytopenia and Paris Trousseau hemorrhagic disease, typical features of this disorder [49]. Moreover, the clinical picture of our patient is complicated by the association of the partial JBS phenotype and an already known pathogenic variant in the PTPN11 gene. RASopathies are a group of autosomal dominant disorders caused by pathogenic variants in genes encoding proteins of the RAS/mitogen-activated protein kinase pathway, such as PTPN11. The clinical spectrum is characterized by specific facial features, congenital heart disease (CHD), and hypertrophic cardiomyopathy (HCM), and variable postnatal growth retardation, neurological involvement, and cancer predisposition [50]. Recently ocular coloboma was assessed in a 7 y.o. patient with a genetically proven Noonan syndrome due to a PTPN11 mutation [51].

A small number of VUS have been also identified, such as a 1.54 Mb deletion in the 16q23.1q23.2 locus. The deletion partially removes WWOX coding region (intron 8–9 to 5′UTR), whose biallelic variants are known to cause a severe early-onset epileptic encephalopathy. No other variants affecting this gene were assessed in our patient (A256). More interesting turns out to be the involvement of the MAF gene, which is found to be completely included in the deletion. Missense mutation in MAF are associated to both autosomal dominant cataract and the Ayme–Gripp syndrome, a clinically homogeneous disorder characterized by congenital cataracts, sensorineural hearing loss, intellectual disability, seizures, brachycephaly, a distinctive flat facial appearance, and reduced growth [52]. Indeed, our patient had no phenotypic overlap with both these conditions and was diagnosed with bilateral aniridia. The MAF bZIP transcription factor (MAF) is an important regulator of eye development, specifically lens development [53]. MAF point mutations have been associated with ocular malformations, such as iris coloboma, congenital cataract, glaucoma, and microphthalmia [54]. To date, MAF whole gene deletions have never been correlated to aniridia. Further experiments needed to be performed to clearly validate this novel association.

Comparing our data with other studies in this field published in the past 5 years, it is evident that the composition of the cohorts analyzed is quite variable, as are the molecular technologies applied. Cross et al. applied both direct sequencing and MLPA in a cohort of 434 subjects undergoing diagnostic testing for PAX6 [55]. Vasilyeva et al., who screened 199 patients for variants at the 11p13 locus, adopted the same analytical approach [56]. In these works, the diagnostic rate is very different, 58.5% and 69%, respectively, and this is due to the different composition of the cohort (59% vs 92% of patients with classical aniridia). When less “narrow” technological approaches were employed, the number of patients analyzed was reduced with a significant decrease in diagnostic yield. Targeted sequencing, WES and WGS were performed with an average of 47% positive molecular diagnoses [11, 30, 57, 58].

In conclusion, our data certainly reiterate the notion that PAX6 alterations are primarily associated with the development of isolated aniridia. It is worth of attention that 20.3% of unique causative variants consist in deletions involving chromosome 11p13, one-third of which exclusively involve PAX6 regulatory regions. The spectrum of deletions identified in this study also enables us to expand the knowledge of how little we still know about the minimal critical region capable of altering eye development. Furthermore, the distinctive enrichment of deletions, most of which involve familial cases, is not due to a marked representation of WAGR cases (only four in the entire cohort) but probably to the size of the cohort itself. Notwithstanding, given the increasing implication of alterations in the regulatory regions of PAX6 eliciting aniridia, a better understanding of PAX6 CREs and of the regulatory regions of other ASD-associated genes would ensure their analysis for diagnostic purposes.

To the best of our knowledge, our cohort is the only Italian cohort published so far. We suggest that the use of WES is critical for the differential diagnosis of those conditions in which there may be phenotypic overlap and in general in ASD, both isolated and syndromic. Indeed, our study has allowed us to identify rare conditions with ocular involvement that contribute to a better understanding of the molecular pathways underlying ocular development.

Lastly, a small percentage of patients still test negative suggesting that: (i) complex rearrangements (i.e., inversions) or deep intronic variants are putatively involved in ocular dysgenesis; (ii) there is room for research into new ultra-rare disease genes.