Skip to main content
PMC home page
Springer logoLink to Springer
. 2018 Feb 19;59(2):151–159. doi: 10.1007/s13353-017-0426-1

The genetics of aniridia — simple things become complicated

Anna Wawrocka 1,✉, Maciej R Krawczynski 1,2
PMCID: PMC5895662  PMID: 29460221

Abstract

Aniridia is a rare, panocular disorder characterized by a variable degree of hypoplasia or the absence of iris tissue associated with additional ocular abnormalities. It is inherited in an autosomal dominant manner, with high penetrance and variable expression even within the same family. In most cases the disease is caused by haploinsufficiency truncating mutations in the PAX6 gene; however, in up to 30% of aniridia patients, disease results from chromosomal rearrangements at the 11p13 region. The aim of this review is to present the clinical and genetic aspects of the disease. Furthermore, we present a molecular diagnostic strategy in the aniridia patients. Recent improvement in the genetic diagnostic approach will precisely diagnosis aniridia patients, which is essential especially for children with aniridia in order to determine the risk of developing a Wilms tumor or neurodevelopmental disorder. Finally, based on the previous studies we describe the current knowledge and latest research findings in the topic of pathogenesis of aniridia and possible future treatment.

Keywords: Aniridia, PAX6, 11p13 microdeletions, MLPA, High-resolution array-CGH

Introduction

Aniridia (OMIM 106210) is a congenital panocular disorder characterized by complete or partial iris hypoplasia presenting in early infancy. Clinical expression is highly variable between families and even in the same family. Foveal hypoplasia resulting in reduced visual acuity, as well as cataract, keratopathy, and glaucoma that sometimes develop in the second or third decade, contribute to visual morbidity (Fig. 1). Visual acuity is variable, it is usually 20/100-20/200, but in some affected individuals it can be better than 20/60 especially when nystagmus is not present (Hingorani et al. 2012). Refractive errors, strabismus and ptosis may also be present in the affected eye (Kokotas and Petersen 2010).

Fig. 1.

Fig. 1

Open in a new tab

A typical phenotype of a complete aniridia with polar posterior cataract (white arrow) and peripheral iris remnants (black arrow)

Some aniridia patients have been reported to present with non-ocular, sensory, and neurological abnormalities. Reduced olfaction seems to be the most common functional deficit (Sisodiya et al. 2001; Hingorani et al. 2012). Neurological abnormalities such as autism and intellectual disability have been described by several authors (Davis et al. 2008; Hu et al. 2015). MRI studies showed irregularities of the anterior commissure, anterior cingulate cortex, cerebellum, temporal and occipital lobes, corpus callosum, pineal gland and olfactory bulb (Sisodiya et al. 2001; Mitchell et al. 2003; Bamiou et al. 2007). Abnormal interhemispheric transfer results in central auditory processing deficits, what can cause hearing difficulties (Bamiou et al. 2007).

Aniridia is an autosomal dominant condition with an incidence of approximately one in 40,000-100,000 live births worldwide (Kokotas and Petersen 2010; Hingorani et al. 2012). About two-thirds of aniridia cases are familial with nearly complete penetrance, the remaining one-third are sporadic and result from de novo mutations (Hingorani et al. 2012). Aniridia can occur either as an isolated malformation or as a part of a syndrome. The best known syndromic form is WAGR syndrome (OMIM 194072) — a contiguous genes deletion syndrome of the 11p13 region (encompassing the PAX6 and WT1 genes). It is characterized by Wilms tumor, aniridia, genitourinary anomalies, and intellectual disability. Patients have 50-70% risk to develop Wilms tumor, which is the most common form of kidney cancer in children. Approximately 30% of patients with the sporadic form of aniridia suffer from this syndrome (Fischbach et al. 2005). Another known form of aniridia is Gillespie syndrome (OMIM 206700) — a rare disorder characterized by non-progressive cerebellar ataxia, intellectual disability, and iris hypoplasia. Gillespie syndrome is estimated to account for about 2% of aniridia-like cases (Gillespie 1965; Ansari et al. 2016). The PAX6 gene mutations were identified in only two individuals described as Gillespie syndrome but with atypical features like corectopia and ptosis (Ticho et al. 2006). In 2016, Gerber et al. identified mutations in the ITPR1 gene in patients affected with Gillespie syndrome (Gerber et al. 2016); however, in most cases the genetic cause of the disease remains unclear.

Structure and function of the PAX6 gene and its protein

The PAX6 gene (OMIM 607108) was the first homeobox gene discovered to play a crucial role in eye development (Glaser et al. 1992). In 1991 it was identified by positional cloning as a causative gene for congenital aniridia (Ton et al. 1991; Glaser et al. 1992).

The PAX6 gene is located on chromosome 11p13, contains 14 exons including an alternatively spliced exon 5a. It is abundantly expressed in the forebrain, early eye structures, ventral spinal cord, and endocrine pancreas (Lauderdale et al. 2000). The PAX6 protein as a transcriptional factor plays a crucial role in embryonic development and organogenesis, including neuro- and oculogenesis (Lang et al. 2007; Kokotas and Petersen 2010). The protein contains two functional domains: paired domain (PD) and homeodomain (HD), separated by the proline/serine/threonine-rich transactivation domain (PST). The paired domain comprises two structurally distinct DNA-binding subdomains: the N-terminal subdomain (NTS) and the C-terminal subdomain (CTS) (Kokotas and Petersen 2010) (Fig. 2). The PAX6 gene produces two alternatively spliced isoforms, with a distinct structure of the PD domain: a 422 amino acid and an alternatively spliced 436 amino acid PAX6 protein (including exon 5a). The insertion, into the NTS subdomain, of 14 additional amino acids encoded by exon 5a abolishes the DNA-binding activity of the NTS and unmasks the DNA-binding ability of the CTS. Thus, exon 5a appears to function as a molecular switch that specifies target genes (Azuma et al. 1999).

Fig. 2.

Fig. 2

Open in a new tab

The PAX6 cDNA, horizontal rectangles represent PAX6 protein domains including: PD-paired domain, LNK-linker region, HD-homeodomain, PST-proline/serine/threonine rich region. Exon 5a is an alternatively spliced exon in the PD domain. The most frequent mutations in the PAX6 gene described in LOVD database are presented above the diagram

Heterozygous PAX6 gene point mutations

To date, more than 400 heterozygous variants of the PAX6 gene have been identified and recorded in the Human PAX6 Allelic Variant Database (Leiden Open Variation Database, LOVD). Of all intragenic point mutations in the PAX6 gene, 94% result in premature termination codon (PTC), C-terminal extensions (CTE) or amino-acid substitutions (missense mutations) (Hingorani et al. 2012). Most of the reported PAX6 gene heterozygous mutations including nonsense and splicing mutations, insertions, and deletions, introduce premature termination codons into the PAX6 open reading frame, resulting in haploinsufficiency. The level of the protein expression from a single functional allele is not sufficient to produce biologically active PAX6 protein (50% reduction in overall activity) (Kokotas and Petersen 2010). The dosage-dependance of the PAX6 gene was supported by studies in naturally occuring rodent models with heterozygous Pax6 mutations (Matsuo et al. 1993; Kokotas and Petersen 2010; Cvekl and Callaerts 2017). Heterozygous mutations in the Pax6 gene cause mouse smalleye (Sey) phenotype demonstrating dosage sensitivity (Hill et al. 1991).

Truncating mutations activate the nonsense-mediated decay (NMD) process, by which mRNA containing premature termination codons are degraded before they produce truncated proteins (Baker and Parker 2004).

C-terminal extension mutations (dominant negative mutations) in the PAX6 gene result from frameshift or point mutations that change the stop codon of the C-terminus to cause run-on translation into 3’UTR. Nonsense-mediated decay is not predicted for CTE transcripts and an extended abnormal protein is produced (Tzoulaki et al. 2005). This type of mutation is usually associated with severe phenotype with pronounced iris hypoplasia and profound visual impairment (Aggarwal et al. 2011).

Single amino acid substitutions (missense mutations) produce a full-length protein that may fold and function abnormally, causing phenotypes ranging from mild iris defects to more severe including optic nerve malformations, Peters anomaly and microphthalmia (Hanson et al. 1994; Grønskov et al. 2001; Azuma et al. 2003).

Heterozygous PAX6 gene mutations account for approximately two-thirds of all aniridia cases (Robinson et al. 2008). The most frequent mutations in the PAX6 gene are present in Fig. 2.

Compound heterozygous PAX6 gene mutations

Families of aniridic parents with heterozygous PAX6 gene mutations were reported in the literature. Affected parents have a 25% risk that they would give birth to a child with both alleles having PAX6 gene mutations. Compound heterozygosity in the PAX6 gene was thought to be lethal, resulting in severe malformations of the fetuses. However, in 1994, Glaser et al. reported the first patient with compound heterozygous PAX6 gene mutations. Two nonsense mutations inherited from the aniridic parents were identified in a newborn with anophthalmia, severe craniofacial, and central nervous system defects. The pattern of malformations was similar to that observed in homozygous Sey mouse (Glaser et al. 1994). Compound heterozygosity was also reported in two fetuses whose parents were affected with aniridia. Both fetuses showed severe brain malformations with increased germinal proliferation, gross disturbances of migration, and organization of the central nervous system (Schmidt-Sidor et al. 2009).

Finally, Solomon et al. reported a patient, who suffered from trisomy 21 and additionally was a compound heterozygote for one missense and one nonsense mutations in the PAX6 gene, inherited from the parents. The presence of both mutations resulted in severe ophthalmological, neurological, and endocrinological disturbances. This four year old patient suffers from microphthalmia, neonatal diabetes mellitus, hypopituitarism, and a complex structural brain anomaly. This was the first reported patient who survived the neonatal period (Solomon et al. 2009).

11p13 microdeletions

In 30% or even in higher proportion of aniridia patients (Wawrocka et al. 2013), the disease results from genomic rearrangements at the 11p13 region including PAX6 whole-gene deletions, microdeletions affecting only the 3′ regulatory enhancers (excluding the PAX6 gene) or contiguous genes deletions of the PAX6 and other neighboring genes, especially WT1 (WAGR syndrome) (Crolla and van Heyningen 2002; Robinson et al. 2008).

Microdeletions comprising the PAX6 gene and the adjacent genes

Microdeletions encompassing the PAX6 and the neighboring genes have been described by several authors (Fantes et al. 1992; Crolla and van Heyningen 2002; Hu et al. 2015). Identified deletions were noted even though the PAX6 gene usually harbors doublecortin domain-containing protein 1 (DCDC1), elongation factor protein (ELP4), brain-derived neurotrophic factor (BDNF), beta polypeptide of follicle-stimulating hormone (FSHB), reticulocalbin 1 (RCN1), partial IMP1 inner-mitochondrial membrane (IMMP1L) (Wawrocka et al. 2013; Hu et al. 2015) and WT1 gene (Crolla and van Heyningen 2002). Sporadic aniridia cases with a deletion of both PAX6 and WT1 genes have been determined in a number of studies (Grønskov et al. 2001; Muto et al. 2002). Deletions including the PAX6 gene and other neighboring genes do not change the clinical expression of the disease in relation to mutation limited only to PAX6. However, for larger deletions encompassing PAX6 and WT1 genes, separated by 700 kb, patients with aniridia represent a more severe phenotype — WAGR syndrome with intellectual disability and high risk of Wilm’s tumor development.

Microdeletions excluding the PAX6 gene — position effect

In 1995, Fantes et al. described two aniridia families with genomic rearrangements at the 11p13 region, that did not include the PAX6 gene sequence and proposed that the causative mechanism of the disease in these families is a “position effect” (Fantes et al. 1995). Thereafter, submicroscopic rearrangements that do not disrupt the PAX6 gene sequence in the aniridia patients were described by several authors. Lauderdale et al. presented two de novo deletions of 11p13, located more than 11 kb downstream of the PAX6 gene. Using human–mouse cell hybrids they showed that the PAX6 gene expression is restricted to normal allele, unambiguously indicating that 3′ regulatory elements are essential for the PAX6 gene expression (Lauderdale et al. 2000). Davis et al. identified in a patient with aniridia, autism and intellectual disability a 1.3 Mb deletion, situated approximately 35 kb downstream of the PAX6 gene, comprising the ELP4, DPH4, DCDC1, DCDC5, MPPED2, and IMMP1L genes (Davis et al. 2008). Genomic rearrangements downstream of the PAX6 gene identified so far in aniridia patients are summarized in Table 1.

Table 1.

Deletions in the 11p13 region, downstream of the PAX6 gene reported previously in the literature

Deleted genes (whole or partially) Distance from the 3′ of PAX6 Genomic coordinates (hg19) Size of deletion Phenotype References
Aniridia or aniridia and other malformations
ELP4, IMMP1L, DNAJC24, DCDC1, DCDC5 22.1 kb ND 975 kb aniridia (Lauderdale et al. 2000)
ELP4, IMMP1L, DNAJC24, DCDC1, DCDC5 11.6 kb ND 1105 kb aniridia
ELP4 5 kb ND ND aniridia, cataracts, glaucoma (D’Elia et al. 2007)
ELP4, IMMP1L, DNAJC24, DCDC1, DCDC5, MPPED2 35 kb chr11:30,448,178-31,802,357 1.3 Mb aniridia, autism, mental retardation (Davis et al. 2008)
ELP4, IMMP1L, DNAJC24, DCDC1 140 kb chr11:31,260,340-31,666,340 406 kb aniridia (Bayrakli et al. 2009)
ELP4, IMMP1L, DNAJC24, DCDC1 123 kb chr11:31,117,827-31,683,687 566 kb aniridia (Cheng et al. 2011)
ELP4, IMMP1L, DNAJC24, DCDC1 1 kb chr11:31,280,628-31,805,329 525 kb aniridia (Zhang et al. 2011)
ELP4, IMMP1L, DNAJC24, DCDC1 85 kb chr11:31,122,161-31,721,030 599-652 kb partial aniridia (Wawrocka et al. 2012)
ELP4 8 kb chr11:31,706,160-31,755,245 bp 49 kb aniridia, developmental delay, a submucous cleft palate, ventriculoseptal defect Simoni et al. 2012
ELP4, IMMP1L, DNAJC24, DCDC1 96 kb chr11:31,118,027-31,710,576 593 kb Rieger anomaly, aniridia (Addis et al. 2015)
ELP4, IMMP1L, DNAJC24, DCDC1 31 kb chr11:31,172,410-31,775,457 603 kb aniridia
ELP4 23 kb chr11:31,605,859-31,783,590 178 kb partial aniridia
ELP4, IMMP1L, DNAJC24, DCDC1, DCDC5 108 kb chr11:30,918,066-31,698,257 780 kb aniridia (Ansari et al. 2016)
ELP4, IMMP1L, DNAJC24, DCDC1, DCDC5 59 kb chr11:31,010,424-31,747,424 737 kb aniridia
ELP4, IMMP1L, DNAJC24, DCDC1 113 kb chr11:31,152,003-31,693,266 541 kb aniridia
ELP4, IMMP1L, DNAJC24 54 kb chr11:31,422,424-31,751,424 329 kb aniridia
ELP4, IMMP1L, DNAJC24, DCDC1 11 kb chr11:31,277,819-31,795,239 517 kb partial aniridia, ataxia, developmental delay
ELP4, IMMP1L, DNAJC24, DCDC1 91 kb chr11:31,147,306-31,714,853 567 kb aniridia (Blanco-Kelly et al. 2017)
ELP4, IMMP1L, DNAJC24, DCDC1 108 kb chr11:31,186,493-31,698,208 512 kb aniridia
ELP4, IMMP1L, DNAJC24, DCDC1 101 kb chr11:31,083,877-31,704,548 620 kb aniridia
Ocular malformations and/or neurodevelopmental disorders without aniridia
ELP4, IMMP1L, DNAJC24, DCDC1, DCDC5 114 kb chr11:31,010,914-31,692,238 681 kb ocular coloboma (Guo et al. 2013)
ELP4, IMMP1L, DNAJC24 191 kb chr11:31,452,082-31,615,319 163 kb intellectual disability, speech abnormalities, autistic behaviors (Balay et al. 2016)
ELP4, IMMP1L, DNAJC24, DCDC1, DCDC5 242 kb chr11:30,991,456-31,564,708 573 kb focal epilepsy with cortical dysplasia, mild developmental delay, ADHD, neurinomas, squint, ptosis, fine motor dyspraxia
ELP4, IMMP1L 260 kb chr11:31,495,260-31,546,276 51 kb cognitive delay, speech and language disorder, reading and spelling disorder, ASD, epilepsy
ELP4 181 kb chr11:31,561,220-31,625,448 64 kb speech and language delay
ELP4 132 kb chr11:31,573,422-31,674,789 101 kb developmental delay, microcephaly
ELP4 164 kb chr11:31,584,329-31,642,325 58 kb language disorder, behavioral problems
ELP4 174 kb chr11:31,601,768-31,632,347 31 kb developmental delay, hypotonia, ventriculomegaly (Addis et al. 2015)
ELP4 84 kb chr11:31,691,270-31,722,740 31 kb developmental delay, speech and language disorder, microcephaly, mild cognitive delay, motor skills development disorder
ELP4 59 kb chr11:31,705,076-31,747,631 43 kb autism, learning difficulties
ELP4 20 kb chr11:31,760,904-31,786,914 26 kb moderate developmental delay, autism
ELP4 4 kb chr11:31,597,322-31,802,120 205 kb severe intellectual disability, muscle hypotrophy, severe dysphagia, craniofacial abnormalities
ELP4 31 kb chr11:31,605,859-31,775,457 170 kb developmental delay, behavioral problems, pervasive developmental disorder
ELP4 31 kb chr11:31,625,389-31,775,457 150 kb behavioral and speech problems, mild mental retardation
ELP4, IMMP1L 151 kb chr11:31,460,506-31,655,108 195 kb autism
ELP4, IMMP1L 199 kb chr11:31,488,890-31,607,986 119 kb autism (Addis et al. 2015)
ELP4, IMMP1L 157 kb chr11:31,518,924-31,649,475 131 kb autism, language delay
ELP4 76 kb chr11:31,576,768-31,653,568 77 kb autism, coordination problems
ELP4 112 kb chr11:31,652,219-31,764,393 112 kb autism, language delay, mild developmental delay, motor delay
Open in a new tab

ND – no data, ASD – autism spectrum disorder, ADHD – attention deficit hyperactivity disorder

Cis-regulatory elements (CREs) such as promoters, enhancers, insulators, and boundary elements play an important role in the regulation of gene expression, they determine the time, place, and the level of the expression of their target genes. Cis-regulatory elements can spread hundreds of kilobases upstream or downstream from the transcription start site of the genes. Human genetic studies using YAC transgenic mouse, DNaseI hypersensitivity mapping and reporter transgenic assays revealed a presence of SIMO enhancer located in the 3′- distal region, 124 kb from the PAX6 polyadenylation site, a retina-specific enhancer located within a fragment containing HS2 and HS3 elements and a lens-specific enhancer. These sequences located within introns of the adjacent ELP4 gene are PAX6-specific long range control elements (DRR – downstream regulatory region) (Kleinjan et al. 2001, 2002). DNaseI hypersensitivity and ultraconservation analysis carried out by McBride et al. allowed identification of new long range enhancers HS5 and HS6 at the Pax6 cis-regulatory region. HS5 plays a role in maintaining the PAX6 gene expression in the optic cup and diencephalon, while HS6 drives the PAX6 gene expression in the developing eye and the precerebellar neuron-epithelium of the hindbrain in a time specific manner during development (McBride et al. 2011). Deletion of the DRR region (including the retina enhancer in the HS2 and HS3 fragment) in mouse abolished Pax6 expression in the retina, iris, and ciliary body indicating that the presence of the DRR region is indispensable in this structure (Kleinjan et al. 2006). Additional cis-regulatory elements may be present in the DRR region, but have not been discovered yet.

Deletion of the SIMO enhancer in mouse did not completely abolish the Pax6 gene expression in the lens due to the presence of additional enhancers regulating its expression in this tissue (Kleinjan et al. 2006). However, Bhatia et al. demonstrated using the BAC-transgenic approach in zebrafish and mouse reporter that continued tissue-specific expression from the Pax6 promoters is critically dependent on the presence of the SIMO enhancer. The point mutation that disrupts the autoregulatory PAX6 binding site within SIMO causes loss of enhancer activity and abolishes lens and late retinal expression in transgenic mouse. The identified mutation of an autoregulatory binding site revealed a distinct regulatory mechanism by disruption of a positive feedback loop critical for gene expression during development (Bhatia et al. 2013).

The previous studies showed that genomic sequences located in a region of approximately 120 kb 3′ to the transcription start site are important for transcriptional activation of the PAX6 gene. Recent studies performed by Ansari et al. in a cohort of aniridia and Gillespie syndrome patients negative for the PAX6 gene mutations suggest a new “critical region” of 244 kb that includes cis-regulatory elements, essential for PAX6 transcriptional activation. This region spans a fragment of the DNAJC24, IMMP1L genes, and introns 1-7 of the ELP4 gene (chr11:31,379,000 (hg18) and 31,622,916 (hg18)) (Ansari et al. 2016).

Microdeletions including or disrupting the ELP4 gene were also shown in patients with neurodevelopmental disorders including autism spectrum, speech/language disorders, epilepsy, and developmental delay. Microdeletions identified by Addis et al. do not involve the entire critical region. Only in two patients with aniridia, deletions downstream of the PAX6 gene including the ELP4 gene affecting the entire critical region were identified (Fig. 3). Therefore, deletions including the critical region cause aniridia or other ocular malformation and may also predispose to neurodevelopmental disorders (Addis et al. 2015).

Fig. 3.

Fig. 3

Open in a new tab

Schematic diagram of the previously reported microdeletions in the 11p13 region, downstream of the PAX6 gene in the aniridia patients. Horizontal blue blocks represent deletions that have been identified in the aniridia patients. The vertical blue dashed lines indicate the “critical region” required for the PAX6 gene transcription suggested by Ansari et al. 2016

PAX6 gene mutations and 11p13 microdeletions in non aniridia patients

Although most mutations in the PAX6 gene are responsible for aniridia, some mutations can be connected to some other ocular disorders including: microphthalmia, microcornea, foveal hypoplasia, ocular coloboma, keratitis, congenital cataract, Gillespie syndrome, Peters anomaly, and morning glory disc anomaly (Mirzayans et al. 1995; van Heyningen and Williamson 2002; Azuma et al. 2003; Tzoulaki et al. 2005; Wang et al. 2012). Aniridia is predominantly associated with truncating mutations in the PAX6 gene, while missense mutations usually lead to non-aniridia phenotypes (Tzoulaki et al. 2005).

Genomic rearrangements at 11p13 region, have also been reported in non aniridia patients, suggesting that clinical manifestation may vary depending on the location of the breakpoints and genes involoved. The 681 kb deletion downstream of the PAX6 gene including the DCDC5, DCDC1, DNAJC24, IMMP1L genes and a part of the ELP4 gene has been discovered in a patient with ocular coloboma (Guo et al. 2013). A familial pericentric inversion of chromosome 11 associated with submicroscopic interstitial deletion of 11p13 and duplication of 11q22.3 was presented by Balay et al. The 11p13 deletion of 163 kb included the IMMP1L gene and part of the DNAJC24 and ELP4 genes. The patient’s phenotype was characterized by intellectual disability, speech abnormalities, and autistic behaviors; however, nobody in this family presented aniridia or other eye anomalies (Balay et al. 2016).

Perspectives of the gene therapy

Identification of the PAX6 gene transcription network, that means genes directly regulated by the PAX6 gene, is essential in explaining tissue sensitivity to PAX6 dosage. Xia Wang et al., using laser capture microdissection in mouse, chromatin immunoprecipitation, promoter-reporter assays, and immunochemistry methods, demonstrated for the first time that the Bmp4, Tgfβ2, and Foxc1 genes are direct downstream targets of Pax6 in developing iris and ciliary body (Wang et al. 2017b). In some patients with either no PAX6 gene intragenic mutations nor 11p13 genomic rearrangements, mutations in the Pax6 downstream targets are the cause of aniridia. Furthermore, the previous study performed by Gregory-Evans et al. demonstrated that by after birth manipulation of Pax6 dosage using nonsense suppression, it is possible to prevent the development of aniridic phenotype in mouse. It suggests that downstream targets are also active after birth. These studies gives an opportunity for future treatment of aniridia using Pax6 targets (Gregory-Evans et al. 2014).

A nonsense suppression therapy is a promising therapeutic approach that has been introduced and successfully used in an animal model. In recent studies, Xia Wang et al. used a nonsense suppression to manipulate Pax6 dosage at different stages during eye development of the small eye mouse model of aniridia. They performed in vivo tests to study effects in the developing retina, cornea, and lens, using Ataluren — a compound with nonsense suppression activity, which suppresses the abnormal nonsense codon in the mRNA template. Eye malformations responded to delivered after birth nonsense supression in a manner dependent on the Pax6 dose and specific time. The obtained results seem to be very promising and give hope for aniridia patients with nonsense mutations. Nonsense mutation supression strategy could be significant for the patients if the drug could be targeted to the affected tissue at the appropriate time (Wang et al. 2017a).

This findings seems to be very important regarding the possible therapeutic startegies for patients with aniridia.

Summary

In many studies it was proven that aniridia is a typical autosomal dominant disorder resulting from inactivation of one allele of the PAX6 gene, predominantly through intragenic mutations leading to premature termination of the protein. Due to the fast development of the molecular diagnostic methods the genetic background of aniridia became much more complex. Chromosomal abnormalities at chromosome 11p13, including the PAX6 gene deletions, contiguous genes deletions of the PAX6 and other neighboring genes, as well as microdeletions affecting only the 3′ regulatory enhancers, have also been reported in association with both sporadic and familial cases of aniridia. The unusual and wide spectrum of molecular defects observed in the aniridia patients makes this disease a model monogenic disorder in terms of its molecular background.

Due to clinical and allelic heterogeneity, as well as the great variability of the genetic mechanisms involved in aniridia, molecular analysis of the patients is performed by combining different molecular approaches including Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), and an array based comparative genomic hybridization (aCGH). Although the intragenic PAX6 gene mutations are more common than microdeletions, in newborns affected with aniridia the analysis toward deletions is recommended first, due to the clinical importance of early WAGR syndrome identification. At the same time this strategy gives an opportunity to detect genomic rearrangements at 11p13 in approximately 30% of aniridia patients. High resolution aCGH, and to some degree also commercially available MLPA set, allow for detection of small intragenic copy number variations (CNVs) at the resolution of single exons (Morice-Picard et al. 2014). Recently, Blanco-Kelly et al. developed a customized single locus aCGH for aniridia called WAGR-array with a high resolution CNV detection and precision of identified breakpoints (Blanco-Kelly et al. 2017). Precise mapping of the breakpoints is very important in accurate molecular diagnosis of children with aniridia in order to determine not only the risk of developing Wilms tumor but also neurodevelopmental disorder (deletion of the ELP4 gene). In situations when MLPA, aCGH, and the PAX6 gene sequencing do not identify any cause of the disease, a whole exome (WES) or whole genome sequencing (WGS) could be used for identification of novel (e.g., deep intronic) mutations in the PAX6 gene or novel genes involved in pathogenesis of aniridia.

In our studies, despite using all the above mentioned techniques, there is still a proportion of aniridia patients that remain unsolved. Our patients do not have the PAX6 gene mutations, nor genomic rearrangements that involve the PAX6 gene or its regulatory elements. It suggests that mutations in the other genes, yet undiscovered genetic loci or unknown mechanisms disrupting the PAX6 gene function could be the cause of aniridia. We await descriptions of both genocopies and possible recessive mechanisms of aniridia that are already suggested by the pedigree analysis.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. Addis L, Ahn JW, Dobson R et al (2015) Microdeletions of ELP4 are associated with language impairment, autism spectrum disorder, and mental retardation. Hum Mutat 36:842–850. 10.1002/humu.22816 [DOI] [PubMed]
  2. Aggarwal S, Jinda W, Limwongse C, et al. Run-on mutation in the PAX6 gene and chorioretinal degeneration in autosomal dominant aniridia. Mol Vis. 2011;17:1305–1309. [PMC free article] [PubMed] [Google Scholar]
  3. Ansari M, Rainger J, Hanson IM, et al. Genetic analysis of “PAX6-negative” individuals with Aniridia or Gillespie syndrome. PLoS One. 2016;11:e0153757. doi: 10.1371/journal.pone.0153757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Azuma N, Yamaguchi Y, Handa H, et al. Missense mutation in the alternative splice region of the PAX6 gene in eye anomalies. Am J Hum Genet. 1999;65:656–663. doi: 10.1086/302529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Azuma N, Yamaguchi Y, Handa H, et al. Mutations of the PAX6 gene detected in patients with a variety of optic-nerve malformations. Am J Hum Genet. 2003;72:1565–1570. doi: 10.1086/375555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Baker KE, Parker R. Nonsense-mediated mRNA decay: terminating erroneous gene expression. Curr Opin Cell Biol. 2004;16:293–299. doi: 10.1016/j.ceb.2004.03.003. [DOI] [PubMed] [Google Scholar]
  7. Balay L, Totten E, Okada L, et al. A familial pericentric inversion of chromosome 11 associated with a microdeletion of 163 kb and microduplication of 288 kb at 11p13 and 11q22.3 without aniridia or eye anomalies. Am J Med Genet A. 2016;170A:202–209. doi: 10.1002/ajmg.a.37388. [DOI] [PubMed] [Google Scholar]
  8. Bamiou D-E, Free SL, Sisodiya SM, et al. Auditory interhemispheric transfer deficits, hearing difficulties, and brain magnetic resonance imaging abnormalities in children with congenital Aniridia due to PAX6 mutations. Arch Pediatr Adolesc Med. 2007;161:463. doi: 10.1001/archpedi.161.5.463. [DOI] [PubMed] [Google Scholar]
  9. Bayrakli F, Guney I, Bayri Y et al (2009) A novel heterozygous deletion within the 3' region of the PAX6 gene causing isolated aniridia in a large family group. J Clin Neurosci. 16(12):1610–4. 10.1016/j.jocn.2009.03.022 [DOI] [PubMed]
  10. Bhatia S, Bengani H, Fish M et al (2013) Disruption of autoregulatory feedback by a mutation in a remote, ultraconserved PAX6 enhancer causes Aniridia. Am J Hum Genet 93:1126–1134. 10.1016/j.ajhg.2013.10.028 [DOI] [PMC free article] [PubMed]
  11. Blanco-Kelly F, Palomares M, Vallespín E, et al. Improving molecular diagnosis of aniridia and WAGR syndrome using customized targeted array-based CGH. PLoS One. 2017;12:e0172363. doi: 10.1371/journal.pone.0172363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cheng F, Song W, Kang Y et al (2011) A 556 kb deletion in the downstream region of the PAX6 gene causes familial aniridia and other eye anomalies in a Chinese family. Mol Vis 10(17)448–55 [PMC free article] [PubMed]
  13. Crolla JA, van Heyningen V. Frequent chromosome aberrations revealed by molecular cytogenetic studies in patients with Aniridia. Am J Hum Genet. 2002;71:1138–1149. doi: 10.1086/344396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Cvekl A, Callaerts P. PAX6: 25th anniversary and more to learn. Exp Eye Res. 2017;156:10–21. doi: 10.1016/j.exer.2016.04.017. [DOI] [PubMed] [Google Scholar]
  15. Davis LK, Meyer KJ, Rudd DS, et al. Pax6 3′ deletion results in aniridia, autism and mental retardation. Hum Genet. 2008;123:371–378. doi: 10.1007/s00439-008-0484-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. D'Elia AV, Pellizzari L, Fabbro D et al (2007) A deletion 3' to the PAX6 gene in familial aniridia cases. Mol Vis 23(13)1245–50 [PubMed]
  17. Fantes JA, Bickmore WA, Fletcher JM, et al. Submicroscopic deletions at the WAGR locus, revealed by nonradioactive in situ hybridization. Am J Hum Genet. 1992;51:1286–1294. [PMC free article] [PubMed] [Google Scholar]
  18. Fantes J, Redeker B, Breen M, et al. Aniridia-associated cytogenetic rearrangements suggest that a position effect may cause the mutant phenotype. Hum Mol Genet. 1995;4:415–422. doi: 10.1093/hmg/4.3.415. [DOI] [PubMed] [Google Scholar]
  19. Fischbach BV, Trout KL, Lewis J, et al. WAGR syndrome: a clinical review of 54 cases. Pediatrics. 2005;116:984–988. doi: 10.1542/peds.2004-0467. [DOI] [PubMed] [Google Scholar]
  20. Gerber S, Alzayady KJ, Burglen L, et al. Recessive and dominant de novo ITPR1 mutations cause Gillespie syndrome. Am J Hum Genet. 2016;98:971–980. doi: 10.1016/j.ajhg.2016.03.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gillespie FD. ANIRIDIA, cerebellar ataxia, and OLIGOPHRENIA in siblings. Arch Ophthalmol (Chicago, Ill 1960) 1965;73:338–341. doi: 10.1001/archopht.1965.00970030340008. [DOI] [PubMed] [Google Scholar]
  22. Glaser T, Walton DS, Maas RL. Genomic structure, evolutionary conservation and aniridia mutations in the human PAX6 gene. Nat Genet. 1992;2:232–239. doi: 10.1038/ng1192-232. [DOI] [PubMed] [Google Scholar]
  23. Glaser T, Jepeal L, Edwards JG, et al. PAX6 gene dosage effect in a family with congenital cataracts, aniridia, anophthalmia and central nervous system defects. Nat Genet. 1994;7:463–471. doi: 10.1038/ng0894-463. [DOI] [PubMed] [Google Scholar]
  24. Gregory-Evans CY, Wang X, Wasan KM, et al. Postnatal manipulation of Pax6 dosage reverses congenital tissue malformation defects. J Clin Invest. 2014;124:111–116. doi: 10.1172/JCI70462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Grønskov K, Olsen JH, Sand A, et al. Population-based risk estimates of Wilms tumor in sporadic aniridia. A comprehensive mutation screening procedure of PAX6 identifies 80% of mutations in aniridia. Hum Genet. 2001;109:11–18. doi: 10.1007/s004390100529. [DOI] [PubMed] [Google Scholar]
  26. Guo H, Dai L, Huang Y, et al. A large novel deletion downstream of PAX6 gene in a Chinese family with ocular Coloboma. PLoS One. 2013;8:e83073. doi: 10.1371/journal.pone.0083073. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hanson IM, Fletcher JM, Jordan T, et al. Mutations at the PAX6 locus are found in heterogeneous anterior segment malformations including Peters’ anomaly. Nat Genet. 1994;6:168–173. doi: 10.1038/ng0294-168. [DOI] [PubMed] [Google Scholar]
  28. van Heyningen V, Williamson KA. PAX6 in sensory development. Hum Mol Genet. 2002;11:1161–1167. doi: 10.1093/hmg/11.10.1161. [DOI] [PubMed] [Google Scholar]
  29. Hill RE, Favor J, Hogan BLM, et al. Mouse small eye results from mutations in a paired-like homeobox-containing gene. Nature. 1991;354:522–525. doi: 10.1038/354522a0. [DOI] [PubMed] [Google Scholar]
  30. Hingorani M, Hanson I, van Heyningen V. Aniridia. Eur J Hum Genet. 2012;20:1011–1017. doi: 10.1038/ejhg.2012.100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Hu P, Meng L, Ma D, et al. A novel 11p13 microdeletion encompassing PAX6 in a Chinese Han family with aniridia, ptosis and mental retardation. Mol Cytogenet. 2015;8:3. doi: 10.1186/s13039-015-0110-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kleinjan DA, Seawright A, Schedl A, et al. Aniridia-associated translocations, DNase hypersensitivity, sequence comparison and transgenic analysis redefine the functional domain of PAX6. Hum Mol Genet. 2001;10:2049–2059. doi: 10.1093/hmg/10.19.2049. [DOI] [PubMed] [Google Scholar]
  33. Kleinjan DA, Seawright A, Elgar G, van Heyningen V. Characterization of a novel gene adjacent to PAX6, revealing synteny conservation with functional significance. Mamm Genome. 2002;13:102–107. doi: 10.1007/s00335-001-3058-y. [DOI] [PubMed] [Google Scholar]
  34. Kleinjan DA, Seawright A, Mella S, et al. Long-range downstream enhancers are essential for Pax6 expression. Dev Biol. 2006;299:563–581. doi: 10.1016/j.ydbio.2006.08.060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Kokotas H, Petersen M. Clinical and molecular aspects of aniridia. Clin Genet. 2010;77:409–420. doi: 10.1111/j.1399-0004.2010.01372.x. [DOI] [PubMed] [Google Scholar]
  36. Lang D, Powell SK, Plummer RS, et al. PAX genes: roles in development, pathophysiology, and cancer. Biochem Pharmacol. 2007;73:1–14. doi: 10.1016/j.bcp.2006.06.024. [DOI] [PubMed] [Google Scholar]
  37. Lauderdale JD, Wilensky JS, Oliver ER, et al. 3′ deletions cause aniridia by preventing PAX6 gene expression. Proc Natl Acad Sci U S A. 2000;97:13755–13759. doi: 10.1073/pnas.240398797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Matsuo T, Osumi-Yamashita N, Noji S, et al. A mutation in the Pax-6 gene in rat small eye is associated with impaired migration of midbrain crest cells. Nat Genet. 1993;3:299–304. doi: 10.1038/ng0493-299. [DOI] [PubMed] [Google Scholar]
  39. McBride DJ, Buckle A, van Heyningen V, Kleinjan DA. DNaseI hypersensitivity and ultraconservation reveal novel, interdependent long-range enhancers at the complex Pax6 cis-regulatory region. PLoS One. 2011;6:e28616. doi: 10.1371/journal.pone.0028616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Mirzayans F, Pearce WG, MacDonald IM, Walter MA. Mutation of the PAX6 gene in patients with autosomal dominant keratitis. Am J Hum Genet. 1995;57:539–548. [PMC free article] [PubMed] [Google Scholar]
  41. Mitchell TN, Free SL, Williamson KA, et al. Polymicrogyria and absence of pineal gland due toPAX6 mutation. Ann Neurol. 2003;53:658–663. doi: 10.1002/ana.10576. [DOI] [PubMed] [Google Scholar]
  42. Morice-Picard F, Lasseaux E, Cailley D, et al. High-resolution array-CGH in patients with oculocutaneous albinism identifies new deletions of the TYR, OCA2, and SLC45A2 genes and a complex rearrangement of the OCA2 gene. Pigment Cell Melanoma Res. 2014;27:59–71. doi: 10.1111/pcmr.12173. [DOI] [PubMed] [Google Scholar]
  43. Muto R, Yamamori S, Ohashi H, Osawa M. Prediction by FISH analysis of the occurrence of Wilms tumor in aniridia patients. Am J Med Genet. 2002;108:285–289. doi: 10.1002/ajmg.10094. [DOI] [PubMed] [Google Scholar]
  44. Robinson DO, Howarth RJ, Williamson KA, et al. Genetic analysis of chromosome 11p13 and thePAX6 gene in a series of 125 cases referred with aniridia. Am J Med Genet Part A. 2008;146A:558–569. doi: 10.1002/ajmg.a.32209. [DOI] [PubMed] [Google Scholar]
  45. Schmidt-Sidor B, Szymańska K, Williamson K, et al. Malformations of the brain in two fetuses with a compound heterozygosity for two PAX6 mutations. Folia Neuropathol. 2009;47:372–382. [PubMed] [Google Scholar]
  46. Simioni M, Vieira TP, Sgardioli IC et al (2012) Insertional translocation of 15q25-q26 into 11p13 and duplication at 8p23.1 characterized by high resolution arrays in a boy with congenital malformations and aniridia. Am J Med Genet A 158A(11):2905–10. 10.1002/ajmg.a.35603 [DOI] [PubMed]
  47. Sisodiya SM, Free SL, Williamson KA, et al. PAX6 haploinsufficiency causes cerebral malformation and olfactory dysfunction in humans. Nat Genet. 2001;28:214–216. doi: 10.1038/90042. [DOI] [PubMed] [Google Scholar]
  48. Solomon BD, Pineda-Alvarez DE, Balog JZ, et al. Compound heterozygosity for mutations in PAX6 in a patient with complex brain anomaly, neonatal diabetes mellitus, and microophthalmia. Am J Med Genet Part A. 2009;149A:2543–2546. doi: 10.1002/ajmg.a.33081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Ticho BH, Hilchie-Schmidt C, Egel RT, et al. Ocular findings in Gillespie-like syndrome: association with a new PAX6 mutation. Ophthalmic Genet. 2006;27:145–149. doi: 10.1080/13816810600976897. [DOI] [PubMed] [Google Scholar]
  50. Ton CC, Hirvonen H, Miwa H, et al. Positional cloning and characterization of a paired box- and homeobox-containing gene from the aniridia region. Cell. 1991;67:1059–1074. doi: 10.1016/0092-8674(91)90284-6. [DOI] [PubMed] [Google Scholar]
  51. Tzoulaki I, White IM, Hanson IM. PAX6 mutations: genotype-phenotype correlations. BMC Genet. 2005;6:27. doi: 10.1186/1471-2156-6-27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wang P, Sun W, Li S, et al. PAX6 mutations identified in 4 of 35 families with microcornea. Invest Ophthalmol Vis Sci. 2012;53:6338–6342. doi: 10.1167/iovs.12-10472. [DOI] [PubMed] [Google Scholar]
  53. Wang X, Gregory-Evans K, Wasan KM, et al. Efficacy of postnatal in vivo nonsense suppression therapy in a Pax6 mouse model of Aniridia. Mol Ther Nucleic Acids. 2017;7:417–428. doi: 10.1016/j.omtn.2017.05.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wang X, Shan X, Gregory-Evans CY. A mouse model of aniridia reveals the in vivo downstream targets of Pax6 driving iris and ciliary body development in the eye. Biochim Biophys Acta - Mol Basis Dis. 2017;1863:60–67. doi: 10.1016/j.bbadis.2016.10.018. [DOI] [PubMed] [Google Scholar]
  55. Wawrocka A, Budny B, Debicki S et al (2012) PAX6 3' deletion in a family with aniridia. Ophthalmic Genet 33(1):44–8. 10.3109/13816810.2011.615076 [DOI] [PubMed]
  56. Wawrocka A, Sikora A, Kuszel L, Krawczynski MR. 11p13 deletions can be more frequent than the PAX6 gene point mutations in polish patients with aniridia. J Appl Genet. 2013;54:345–351. doi: 10.1007/s13353-013-0154-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Zhang X, Zhang Q, Tong Y, et al. Large novel deletions detected in Chinese families with aniridia: correlation between genotype and phenotype. Mol Vis. 2011;17:548–557. [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Applied Genetics are provided here courtesy of Springer

RESOURCES