Molecular Vision 2007; 13:1245-1250 <>
Received 19 April 2007 | Accepted 19 July 2007 | Published 23 July 2007

A deletion 3' to the PAX6 gene in familial aniridia cases

Angela Valentina D'Elia,1 Lucia Pellizzari,2 Dora Fabbro,2 Annalisa Pianta,1 Maria Teresa Divizia,3 Rosanna Rinaldi,4 Barbara Grammatico,4 Paola Grammatico,4 Carlo Arduino,5 Giuseppe Damante1

1Dipartimento di Scienze e Tecnologie Biomediche, Università di Udine and 2Azienda Ospedaliero-Universitaria Santa Maria della Misericordia, Udine, Italy; 3Genetica Molecolare, Istituto Gaslini, Genova, Italy; 4Genetica Medica, "Sapienza - Università di Roma", Azienda Ospedaliera S. Camillo- Forlanini, Roma, Italy; 5Genetica Medica, Azienda Ospedaliera S. Giovanni Battista, Torino, Italy

Correspondence to: Professor Giuseppe Damante, Dipartimento di Scienze e Tecnologie Biomediche, Piazzale Kolbe 1 - 33100, Udine, Italy; Phone: +39 0432 494374; FAX: +39 0432 494379; email:


Purpose: PAX6 mutations cause aniridia as well as other various congenital eye abnormalities. Aniridia can be due to both point mutations and chromosomal deletions/rearrangements. Therefore, a complete search for PAX6 gene alterations in aniridia subjects requires a technically complex approach involving the comprehension of fluorescence in situ hybridization (FISH) analysis. In the present study, an Italian casistic of aniridia patients has been investigated and a quantitative polymerase chain reaction (PCR) assay to detect PAX6 gene deletions was set up.

Methods: Twenty-one aniridia patients were screened for point mutations (missense, nonsense, splicing-affecting, and short insertion/deletion) by using single-stranded conformational polymorphism (SSCP) and denaturing high performance liquid chromatograpy (dHPLC). To reveal deletions not detectable by SSCP or dHPLC, a quantitative PCR approach was set up for the PAX6 structural gene and for regions 5' and 3' to it at the level of WT1 and ELP4, respectively.

Results: Point mutations were found in 7 out of 21 patients. Three out of twenty-one patients showed deletions at the level of the PAX6 structural gene. In addition, two familial cases showed an undamaged PAX6 gene but a deletion in the region 3' to it at level of the ELP4 gene. In one of the families, the presence of the deletion has been confirmed by linkage analysis of polymorphic markers.

Conclusions: In our casistic, a significant fraction of familial aniridia patients appears to be caused by a 3' deletion to PAX6, suggesting that evaluation of this alteration should be included in routine procedures of aniridia patients analysis. The quantitative PCR assay described here represents a simple approach to accomplish this task.


The PAX gene family encodes for transcription factors that are essential for the development of various tissues. PAX6 encodes for a protein that recognizes specific DNA sequences by two highly conserved DNA-binding domains (the paired domain and the homeodomain) [1,2] and controls development of the eye, pancreas, and brain. In the developing eye, PAX6 is expressed in multiple tissues including the lens and neuroretina. In humans, heterozygous PAX6 mutations cause aniridia as well as other various congenital eye abnormalities such as Peters' anomaly, iris hypoplasia, corneal opacification, congenital cataracts, and glaucoma. In mice and rats, Pax6 mutations are responsible for the Small eye (Sey) phenotype [3].

Aniridia can be due to six different categories of PAX6 mutations including nonsense, splicing, frame-shifting insertions or deletions, in-frame insertions or deletions, missense, and run-on mutations [4]. Large chromosomal rearrangements can also be a cause of aniridia [5]. A significant proportion of chromosomal abnormalities consists in submicroscopic deletions, which have been observed by using a complex fluorescence in situ hybridization (FISH) approach [6]. In addition to mutations located inside PAX6, sporadic aniridia cases have been described with deletions not involving the structural gene but involving the removal of elements with transcriptional functions [7]. In the present study, an Italian cohort of aniridia patients has been investigated. By using SSCP and dHPLC as well as a quantitative real-time PCR approach, several mutations were identified. For the first time, we show that a 3' deletion to PAX6, associated with the apparent integrity of the structural gene, occurs in a significant proportion of familial aniridia cases. A quantitative PCR approach to detect deletions in PAX6 and in neighboring regions is presented here.



From 2001 to 2006, a total of 21 Italian aniridia patients were enrolled for this study. In nine patients, aniridia was familial since affected subjects were present in two or more generations. After informed consent was obtained from all patients, samples of 5 ml blood from peripheral veins were collected and genomic DNA from isolated leukocytes was prepared using the Gentra Blood kit. Sixty normal volunteers were used as control subjects. In order to set up quantitative PCR to detect deletions of PAX6, three aniridia patients with known PAX6 gene deletions were used (two of them were kindly provided by V. van Heyningen) [8,9].

Point mutation and deletion analysis

The presence of point mutations in PAX6 was evaluated both by SSCP and dHPLC according to standard protocols [10]. The presence of deletions within PAX6 (at levels of exons 1 and 13), 5' to PAX6 (at the level of WT1), and 3' to PAX6 (at level of ELP4, near the D11S995 locus) was evaluated by quantitative real-time PCR by using a TaqMan system with an ABI 7300 instrument. Assays were set up by using aniridia patients with known deletions of PAX6. Each assay was performed by using 10-20 ng of genomic DNA. The sequences of oligonucleotides that have been utilized are shown in Table 1. PCR was done in a final volume of 12.5 μl using the Platinum Quantitative PCR SuperMix- UDG with Rox (Invitrogen, S. Giuliano Milanese, Italy). Each assay was done in triplicate. The measure of the RNAseP gene (Assay on Demand; Applied Biosystems, Monza, Italy) was used to normalize each value.

Linkage analysis

Linkage analysis in family A13 was performed by investigating the following microsatellites: PAX6 CA/GT, WT1.PCR2, GDB:250586, and D11S995. The location of these markers with respect to WT1, PAX6, and ELP4 is shown in Figure 1. Oligonucleotides utilized for amplification are indicated in Table 1. The PCR amplification parameters were denaturation at 95 °C for 45 s; annealing at 55 °C for 60 s; and elongation at 72 °C for 60 s; 30 cycles. PCR products were labeled by using 32Pα-dCTP. The size of the alleles was determined by running PCR products on denaturing polyacrilamide gel electrophoresis and autoradiography of the gels.


A cohort of 21 aniridia patients has been investigated. The presence of point mutations (missense, nonsense, splicing-affecting, and short insertion/deletion) in PAX6 was investigated by using SSCP and dHPLC. A total of seven point mutations (33%) were identified (Table 2). Four of them are present in the PAX6 mutation database; the remaining (W257X, 738_739insAG and c.472del8) are not present. Only one missense mutation was found (c.719C>G) in which serine is changed to arginine in the COOH subdomain of the paired domain (S119R in Table 2).

Globally, in our casistic, point mutations were identified only in a small fraction of patients. Since submicroscopic deletions are quite common in the human genome [11], we set up a quantitative real-time PCR approach to detect the presence of this kind of mutation within and nearby PAX6. Taqman systems were generated to detect deletions at the 5' and 3' ends of PAX6 encoding sequence (exons 1 and 13, respectively) as well as deletions at the WT1 and ELP4 gene regions, which are located at the 5' and 3' of PAX6, respectively (Figure 1). The single copy RNaseP gene was chosen to normalize PAX6 gene values. Assays for exons 1 and 13 of PAX6 were set up by using subjects with known gene deletions. Examples of amplification plots are shown in Figure 2A. As expected, heterozygous subjects for the PAX6 deletion show half values with respect to normal controls (Figure 2B). Thus, we used this approach to investigate our casistic. As shown in Table 2, the PAX6 deletion was found in three patients. In patient A1, the deletion appears relatively large, including also the WT1 gene. Instead, the deletion detected in patient A9 is limited to only part of the PAX6 structural gene. Deletion in the ELP4 gene region, not involving the PAX6 structural gene, was present in two familial cases (A13 and A16 in Table 2). An example of amplification plot showing ELP4 gene deletion is shown in Figure 3A. In both families, A13 and A16, the ELP4 gene deletion was present in all subjects with aniridia but not in the investigated normal relatives (Figure 3B). In both families, the deletion was not detectable by conventional cytogenetic analysis (data not shown). All affected patients of family A13 presented bilateral aniridia associated with bilateral cataract and glaucoma. In family A16, all affected patients showed bilateral aniridia; the oldest patient had cataract in the left eye and one of her daughters presented bilateral cataract. No case of glaucoma was reported in family A16. Therefore, patients having deletion 3' to PAX6 exhibit no significant phenotypic differences with respect to patients with point mutations.

The deletion in ELP4 gene region was never detected in a cohort of 60 control subjects, indicating that it may be a pathological mutation.

In order to verify the presence of a deletion at the level of ELP4, we used an approach different from quantitative PCR, the familial cases provided us with the opportunity to search for gene deletion by using polymorphic markers (microsatellites). The microsatellites that were investigated and their positions are shown in Figure 1. In the case of the ELP4 gene region, a marker located very close to the amplicon investigated in quantitative PCR was utilized (Figure 1). The linkage analysis approach was highly informative for family A13 (Figure 4), in which subject II-2 and subject III-2 clearly display "loss of heterozygosis" at marker D11S995, which is located inside the ELP4 gene but not at markers GDB:250586 and WT1.PCR2, which are located inside the WT1 gene, and PAX6 CA/GT, which is located inside PAX6.


In the present investigation we report PAX6 gene mutations found in Italian aniridia subjects. Out of 21 patients, SSCP and dHPLC analysis revealed seven point mutations consisting of two splicing-affecting mutations, two indels, two nonsense mutations, and one missense mutation. This latter introduces arginine for serine at residue 119 of the protein. This change is located in a paired domain, which represents one of the two DNA-binding domains of PAX6. However, gel-retardation assays indicate that this mutation does not modify the PAX6 DNA binding properties (unpublished data). Since the paired domain in addition to DNA binding is able to establish interactions with other proteins [12], our data may suggest that S119R causes aniridia through modifications of the protein-protein interactions established by the paired domain.

The major finding of the present study is the observation that a significant proportion of aniridia cases are associated with deletions in the ELP4 gene region and not involving the PAX6 structural gene. Sporadic aniridia cases due to submicroscopic deletions of the region 3' to PAX6 have been already reported along with data indicating no PAX6 expression in the allele harboring the deletion [7]. To our knowledge, familial cases with this alteration are reported for the first time. The 3' deletion should disrupt the function of a putative PAX6 enhancer. In fact, DNAse hypersensitivity sites and PAX6 transcriptional control regions have been identified in this region [13].

Accordingly, in a recent study, using a whole-genome comparison between humans and the pufferfish Fugu rubripens as well as in vivo assay with zebrafish embryos, a highly-conserved functional element in the context of the ELP4 gene has been identified [14]. In addition, it has been recently shown that the absence of distal regulatory elements abolishes PAX6 expression in specific tissues despite the presence of more proximal enhancers [15].

Another interesting point of this study consists in the finding of an aniridia case due to a partial PAX6 gene deletion. In fact, subject A9 shows deletion only in the 3' of the PAX6 structural gene without apparent involvement of the ELP4 gene region. However, it should be pointed out that our data do not rule out involvement of the ELP4 gene region. In fact, the ELP4 primers that we have used amplify sequences 235 kb away from PAX6 (Figure 1). Therefore, it is possible that the deletion involves the ELP4 gene region nearest to PAX6.

Submicroscopic copy number variations are quite common in the human genome [11]; thus, the concept of the copy number polymorphism is emerging. Submicroscopic deletions may play a role in human diseases either by disrupting coding sequences or by loss of gene expression regulatory elements. Our data support the notion that efforts should be made to include the search for submicroscopic deletions in routine protocols for aniridia molecular diagnosis. By definition, submicroscopic deletions are not detected by conventional cytogenetic analysis. Molecular cytogenetic approaches such as FISH [6] require living cells and are relatively time-consuming. The quantitative real-time PCR that we have set up is very simple, requires only a small amount of genomic DNA, thus, challenges eventual MLPA (multiplex ligation-dependent probe amplication)-based approaches [16]. Our approach comprehends also the analysis of the presence of WT1 gene deletions, enabling the ability to assess the risk for Wilms tumor.


This work is funded by grants from MIUR (PRIN n deg 2005060778_002) and Regione Friuli Venezia Giulia to GD. We thank Veronica van Heyningen for providing positive controls to set up a quantitative PCR as well as for additional help and suggestions.


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