Molecular Vision 2006; 12:318-323 <>
Received 19 January 2006 | Accepted 6 April 2006 | Published 7 April 2006

PAX6 gene intragenic deletions in Mexican patients with congenital aniridia

Arturo Ramirez-Miranda, Juan Carlos Zenteno

Research Unit and Department of Genetics, Institute of Ophthalmology "Conde de Valenciana", Mexico City, Mexico

Correspondence to: Juan Carlos Zenteno, MD, PhD; Unidad de Investigación, Instituto de Oftalmología "Conde de Valenciana", Chimalpopoca 14, Col. Obrera, México City, 06800, México; Phone: (5255) 55 88 46 00 ext 212; FAX: (5255) 55 88 46 00 ext 214; email:


Purpose: To present the results of molecular analysis of the PAX6 gene in a group of patients with congenital aniridia from Mexican mestizo origin, a previously unstudied ethnic group.

Methods: Five unrelated affected probands, four pertaining to familial cases and one sporadic, were studied at the Institute of Ophthalmology "Conde de Valenciana" in Mexico City. All patients underwent full ophthalmologic examination as well as PAX6 analysis in genomic DNA using a combination of exon-by-exon PCR amplification, direct sequencing, and allele-specific cloning/sequencing. Available affected relatives were also investigated.

Results: Three novel intragenic deletions were identified: a 15 bp deletion in exon nine that removes the last two codons of the exon and the first nine bases of intron 10, including the conserved GT splicing donor signal; a 14 bp deletion in exon six that introduces a premature stop signal 15 codons downstream and a four bp deletion in exon seven, which introduces a stop signal 22 codons downstream, in three unrelated probands. Although unrelated, these three latter cases came from the same geographical area, strongly suggesting a founder mutation effect as the source of the anomaly.

Conclusions: Our study provides the first molecular analysis of the PAX6 gene in Mexican subjects with congenital aniridia, identifies three novel intragenic PAX6 deletions, and suggests the occurrence of a PAX6 founder mutation effect in this population. Our results also confirm the current notion that PAX6 truncating mutations are overwhelmingly associated with aniridia regardless of their location in the gene.


Eye morphogenesis in humans involves a molecular genetic cascade in which a number of developmental genes interact in a highly organized process during the embryonic period to produce functional ocular structures [1]. Among these genes, PAX6 has an essential role as it encodes a phylogenetically conserved transcription factor almost universally employed for eye formation in animals with bilateral symmetry, despite widely different embryological origins [2]. To direct eye development, PAX6 regulates the tissue-specific expression of diverse molecules, including transcription factors, cell adhesion molecules, hormones, and structural proteins [3]. For example, during the early stages of eye development, PAX6 induces the differentiation of progenitor cells into neurons in the retina, as well as the expression of crystallins in lens epithelial cells [4]. In humans, PAX6 is located on chromosome 11p13, and its mutations lead to a variety of hereditary ocular malformations of the anterior and posterior segment that includes aniridia [5], coloboma of the iris [6], keratitis [7], congenital cataracts [8], Peter's anomaly [9], and optic nerve defects [10].

Aniridia is a congenital severe ocular malformation that has an incidence of approximately one in 60,000-100,000 live births. It is inherited in an autosomal dominant fashion with high penetrance and variable expression [11]. To date, more than 250 PAX6 mutations have been identified in aniridic subjects worldwide, with point nonsense mutations being the more common type of change [12]. In an excellent recent review by Tzoulaki et al. [12], it was clearly recognized that the aniridia phenotype is predominantly associated with mutations that introduce a premature termination codon, while nonaniridia phenotypes are mostly due to missense mutations [12]. Frame-shifting insertions or deletions were the second most common type of lesion accounting for 25% of all aniridia-associated mutations identified in PAX6.

In the present study, we report the molecular analysis of PAX6 in five Mexican aniridia cases (four familial and one sporadic) in which three different novel PAX6 intragenic deletions were identified. Even though PAX6 mutations have been reported in various racial groups, this is the first study in the Mexican mestizo population and our results suggest a probable different mutational spectrum, including a founder mutation effect, in this ethnic group.


A total of nine affected individuals, eight pertaining to four familial cases and one sporadic, were studied at the Institute of Ophthalmology "Conde de Valenciana" in Mexico City. All patients underwent full ophthalmologic examination including visual acuity, slit lamp inspection, intraocular pressure measurement by applanation tonometry, and funduscopic evaluation.

Family 1 (Patients 1-3)

This family included three affected individuals in two generations: a father aged 26 years (patient 1), a nine-year-old daughter (patient 2), and a seven-year-old son (patient 3). All showed a similar phenotype characterized by bilateral aniridia with no iris remnants, normal sized corneas with mild epithelial keratitis, and oscillatory nystagmus. For all three subjects, visual acuity was 20/200 and intraocular pressure was within normal limits. No abnormalities were detected in the posterior segment. Patient 1 had a bilateral nuclear cataract while patient 3 presented with bilateral polar cataracts.

Patient 4

This sporadic case was an eight-year-old girl who exhibited total bilateral aniridia (with no evidence of iris remnants), oscillatory nystagmus, and bilateral nuclear cataracts. Both corneas were of normal size and without opacities. At her first evaluation, she demonstrated visual acuity of 20/200, intraocular pressure of 38 mm Hg, and bilateral nuclear cataracts. There were incipient glaucomatous changes in the optic cup, and she started treatment for glaucoma. No other funduscopic abnormalities were evident. Both her parents were unaffected.

Family 2 (Patients 5 and 6)

This family came from the central region of Mexico. A mother aged 39 years (patient 5) and her 11-year-old daughter (patient 6) presented with bilateral aniridia without iris remnants and visual acuity of 20/200. Patient 5 had normal sized corneas with severe progressive epithelial keratitis (Figure 1) while patient 6 had normal sized and transparent corneas. Both individuals had transparent lenses, normal posterior segment, and intraocular pressure within the normal range. A head CT Scan in patient 5 demonstrated right atrophic temporal lobe and ipsilateral hypertrophic sphenoid sinuses. The occurrence of affected subjects in previous generations was unknown.

Family 3 (Patients 7 and 8)

This family also came from central Mexico. Two affected individuals presented: a 40-year-old female (patient 7) and her 12-year-old son (patient 8). The mother had bilateral coloboma of the iris (Figure 2), visual acuity of 20/20 in each eye, formed anterior chamber, transparent corneas and lenses, normal intraocular pressure, and no pathologic funduscopic features. In contrast, her son showed bilateral total aniridia without evidence of iris remnants (Figure 2), visual acuity of 20/25 in each eye, oscillatory nystagmus, normal sized and transparent corneas, and incipient nuclear cataracts in both lenses. Intraocular pressure was within normal limits, and funduscopic examination did not reveal anomalies in the retina. The occurrence of affected family members in previous generations was not confirmed.

Patient 9

This 38-year-old male presented with total bilateral aniridia, oscillatory nystagmus, bilateral nuclear cataract, normal sized and transparent corneas, 20/200 visual acuity, and normal appearing retina. The patient has three brothers and three sons with the disease but none were available for the study. Previous familial history was not available but the patient's paternal family came from the central region of Mexico.

Molecular methods

After obtaining local Ethics Committee approval and informed consent from patients or their parents, blood samples were drawn by venipuncture, and genomic DNA was extracted using the Puregene DNA Purification Kit (Gentra Systems, Minneapolis, MN). PCR amplification of the complete coding region of the PAX6 gene (14 exons) was achieved using the pairs of primers and temperatures listed in Table 1. Each 25 μl PCR amplification reaction contained 1X buffer, 150 ng of genomic DNA, 0.2 mM of each dNTP, 2U Taq polymerase, 1 mM of forward and reverse primers, and 1.5 mM MgCl2. PCR products were analyzed in 1.5% agarose gels from which the bands with the amplified templates were excised, and the DNA subsequently purified with the help of the Qiaex II kit (Qiagen, Hilden, Germany). Direct automated sequencing of PAX6 was performed with the BigDye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA), adding about 15 ng of template DNA in each reaction and using a temperature program that included 25 cycles of denaturation at 97 °C for 30 s, annealing at 50 °C for 15 s, and extension at 60 °C for 4 min. Samples were analyzed in an ABI Prism 310 Genetic Analyzer (Applied Biosystems). For allele-specific cloning of the PCR amplicons displaying mutations in direct sequencing, new products were amplified from genomic DNA, gel-purified, ligated by means of a TA-ligation method into the TA-cloning vector pGEM-T (Promega, Madison, WI), and subcloned into DH5α E. coli competent cells (Invitrogen, Carlsbad, CA) using standard procedures. The plasmid inserts were sequenced with the protocol previously described and using the forward pUC/M13 primer. Wild-type and mutated sequences were compared manually.


Direct sequencing of PAX6 in the five probands showed a pattern of peak overlap characteristic of superimposed sequences due to heterozygosity for insertion/deletion events. Allele-specific sequencing of the patient's PAX6 subcloned products demonstrated short intragenic deletions in all subjects. Patient 1 had a deletion of 15 bp in exon nine, which eliminates the last six nucleotides of this exon (c.1122-1127delATACAG, nucleotide numbering according to the Human PAX6 Allelic Variant Database) and the initial nine bases on intron 10, including the conserved GT splicing donor signal (Figure 3). Patient 4 exhibited a 14 bp deletion in exon six (c.575-588delCGGTGGTAGTAAAC), which originates a frame-shifting mutation by introducing a premature stop signal (TAA) 15 codons downstream (Figure 4). Interestingly, unrelated patients 5, 7, and 9 showed a same deletion of four bp in exon seven (c.732-735delAACA), which originates a frame-shifting and introduces a stop signal (TAA) 22 codons downstream (Figure 5). Mutations were confirmed by sequencing both sense and antisense strands in the propositi and in all available affected relatives (except in patients 4 and 9). In each case, the normal and abnormal allele were identified after sequencing the subcloned PCR products. Control sequences from ligated/subcloned PAX6 exon seven from normal subjects always demonstrated normal alleles. The parents of sporadic patient 4 exhibited normal PAX6 sequences. Affected relatives of patient 9 were not available for analysis.


PAX6 is a highly conserved transcription factor essential for the development of the eyes in vertebrate and invertebrate species. To date, more than 250 mutations have been identified in subjects with congenital aniridia and at least 29 others in patients with diverse ocular phenotypes as iris coloboma, keratitis, Peter's anomaly, and optic nerve anomalies [12]. Aniridia-causing frame-shifting insertions or deletions account for 25% of all gene lesions described so far in PAX6. In this paper, we describe the identification of three novel intragenic PAX6 deletions, including a recurrent four bp deletion recognized in three apparently unrelated families. Interestingly, however, these three families originate from the same geographical (central) area of the country, strongly suggesting a founder mutation effect. To the best of our knowledge, this is the unique report of a probable founder effect in a PAX6 recurrent mutation.

This is the first study of the PAX6 status in Mexican aniridic patients. Although the sample is small, our results suggest that this population may have a different PAX6 mutational spectrum with a high proportion of deletion mutations. Frame-shifting mutations causing aniridia have been predominantly identified in PAX6 exons six, five, and seven, which is in agreement with our results that show deletions in exons six and seven. The recurrent four bp deletion detected in exon seven has not been previously described, and the fact that patients harboring this deletion originated from the same geographical area in our country suggests a founder mutation effect. The lack of familial information regarding previous generations makes it difficult to trace affected ancestors and to uncover common relatives between these families.

However, another possibility is that these cases could represent independent mutational events leading to a recurrent mutation. In this sense, it is interesting to consider the report of an identical four bp AACA deletion arising independently in the iduronate-2-sulphatase gene in unrelated patients with mucopolysaccharidosis type II (Hunter syndrome) [13]. These recurrent deletions could be related to a specific sequence-directed mutagenesis mechanism. One hypothesis is that direct repeats (two bp or more) flanking or overlapping the locus, or both, may cause slipped mispairing and eventually loop out a small segment of nucleotides [14,15].

Most affected subjects in our series (8/9) exhibited congenital bilateral aniridia, which is in agreement with the strict genotype-phenotype correlation identified in PAX6 mutations in which frame-shifting mutations are almost invariably associated with absence of the iris [12]. However, patient 7 (from family 3) presented bilateral coloboma of iris, a phenotype exclusively associated with missense point mutations [12,16]. Notably, the eight-year-old son of this patient exhibited bilateral total aniridia, providing another example of intrafamilial clinical heterogeneity associated to a particular PAX6 mutation [6,17,18]. Modifier (yet unknown) genes are the probable source for this clinical variability.

The deletions identified in this study affect the paired domain (partially encoded by exon six), the linker region (exon seven), and the homeodomain region (exon nine). As mentioned, independent of the location of the mutation, our patients exhibited an aniridia phenotype, reinforcing the current notion that truncating mutations are overwhelmingly associated with aniridia regardless of their location in the gene [12]. It is interesting to note that the 15 bp deletion detected in exon nine eliminates the last six bases of that exon as well as the nine first nucleotides of intron 10, including the conserved GT splicing donor site. It is predicted that translation will continue until a new GT site is read, and it occurs three codons downstream. These triplets coding for aspartic acid, cysteine, and alanine are read from the intron 10 sequence (Figure 3, bottom).

PAX6 is also required for normal development of many regions of the central nervous system, including the mammalian forebrain, hindbrain, and spinal cord [19]. Several brain malformations have been identified in patients with PAX6 mutations including absence/hypoplasia of the anterior commissure [20], reduction in the callosal area [21], and polymicrogyria and absence of pineal gland [22]. In our study, a patient harboring the four bp deletion in exon seven of PAX6 presented atrophic right temporal lobe and ipsilateral hypertrophic sphenoid sinuses. These structural anomalies are features of the Dyke-Davidoff-Masson syndrome, a condition radiologically characterized by variable degrees of unilateral loss of cerebral volume and associated compensatory bone alterations in the calvarium, like thickening, hyperpneumatization of the paranasal sinuses, and mastoid cells [23]. However, our patient has no evidence of neurological signs or mental impairment, consistent clinical features of this disorder. Unfortunately, cerebral CT scans in other patients carrying the same four bp deletion were not performed.

All mutations identified in our patients are predicted to generate truncated PAX6 polypeptides lacking the transactivation domain. To avoid the potentially lethal consequences of producing truncated polypeptides which could interfere with vital cell functions, mRNAs carrying premature termination codons are rapidly degraded in vivo by a form of RNA surveillance known as nonsense-mediated mRNA decay (NMD) [24]. Recent experimental evidence indicates that PAX6 proteins with COOH-terminal deletions exert strong dominant negative activity by binding to target DNA sequences without activating downstream genes and potentially interfering with the function of the normal PAX6 protein [25,26]. Consequently, it might be expected that naturally occurring truncating mutations after the homeodomain might have dominant negative activity and that they result in phenotypes more severe than, or obviously different from, truncating mutations in the DNA binding regions. If NMD, however, acts on truncated PAX6 proteins in vivo, all alleles containing premature termination codons will be null alleles, and no phenotypic differences should be observed. The fact that virtually all truncating mutations in PAX6 result in the clinical spectrum of aniridia suggests that NMD is a major mechanism acting on PAX6 mutant alleles, and consequently that most truncated proteins are unlikely to be produced at significant levels and therefore unable to exert dominant negative effects [12].

In conclusion, our study provides the first molecular analysis of the PAX6 gene in Mexican subjects with congenital aniridia, identifies three novel intragenic PAX6 deletions, and suggests for the first time the occurrence of a PAX6 founder mutation effect.


The authors thank the "Conde de Valenciana Foundation" patronage for financial support and also Dr. Gerardo Gascon-Guzman and Dr. Roberto Velazquez-Montoya for referral and clinical assessment of patients.


1. Graw J. The genetic and molecular basis of congenital eye defects. Nat Rev Genet 2003; 4:876-88.

2. Nilsson DE. Eye evolution: a question of genetic promiscuity. Curr Opin Neurobiol 2004; 14:407-14.

3. Simpson TI, Price DJ. Pax6; a pleiotropic player in development. Bioessays 2002; 24:1041-51.

4. Ashery-Padan R, Gruss P. Pax6 lights-up the way for eye development. Curr Opin Cell Biol 2001; 13:706-14.

5. Davis A, Cowell JK. Mutations in the PAX6 gene in patients with hereditary aniridia. Hum Mol Genet 1993; 2:2093-7.

6. Vincent MC, Gallai R, Olivier D, Speeg-Schatz C, Flament J, Calvas P, Dollfus H. Variable phenotype related to a novel PAX 6 mutation (IVS4+5G>C) in a family presenting congenital nystagmus and foveal hypoplasia. Am J Ophthalmol 2004; 138:1016-21.

7. Mirzayans F, Pearce WG, MacDonald IM, Walter MA. Mutation of the PAX6 gene in patients with autosomal dominant keratitis. Am J Hum Genet 1995; 57:539-48.

8. Glaser T, Jepeal L, Edwards JG, Young SR, Favor J, Maas RL. PAX6 gene dosage effect in a family with congenital cataracts, aniridia, anophthalmia and central nervous system defects. Nat Genet 1994; 7:463-71. Erratum in: Nat Genet 1994; 8:203.

9. Hanson IM, Fletcher JM, Jordan T, Brown A, Taylor D, Adams RJ, Punnett HH, van Heyningen V. Mutations at the PAX6 locus are found in heterogeneous anterior segment malformations including Peters' anomaly. Nat Genet 1994; 6:168-73.

10. Azuma N, Yamaguchi Y, Handa H, Tadokoro K, Asaka A, Kawase E, Yamada M. Mutations of the PAX6 gene detected in patients with a variety of optic-nerve malformations. Am J Hum Genet 2003; 72:1565-70.

11. Glaser T, Walton DS, Maas RL. Genomic structure, evolutionary conservation and aniridia mutations in the human PAX6 gene. Nat Genet 1992; 2:232-9.

12. Tzoulaki I, White IM, Hanson IM. PAX6 mutations: genotype-phenotype correlations. BMC Genet 2005; 6:27.

13. Li P, Bellows AB, Thompson JN. Molecular basis of iduronate-2-sulphatase gene mutations in patients with mucopolysaccharidosis type II (Hunter syndrome). J Med Genet 1999; 36:21-7.

14. Flanagan JG, Lefranc MP, Rabbitts TH. Mechanisms of divergence and convergence of the human immunoglobulin alpha 1 and alpha 2 constant region gene sequences. Cell 1984; 36:681-8.

15. Vilchis F, Ramos L, Kofman-Alfaro S, Zenteno JC, Mendez JP, Chavez B. Extreme androgen resistance in a kindred with a novel insertion/deletion mutation in exon 5 of the androgen receptor gene. J Hum Genet 2003; 48:346-51.

16. Hanson IM. PAX6 and congenital eye malformations. Pediatr Res 2003; 54:791-6.

17. Sale MM, Craig JE, Charlesworth JC, FitzGerald LM, Hanson IM, Dickinson JL, Matthews SJ, Heyningen Vv V, Fingert JH, Mackey DA. Broad phenotypic variability in a single pedigree with a novel 1410delC mutation in the PST domain of the PAX6 gene. Hum Mutat 2002; 20:322.

18. De Becker I, Walter M, Noel LP. Phenotypic variations in patients with a 1630 A>T point mutation in the PAX6 gene. Can J Ophthalmol 2004; 39:272-8.

19. Walther C, Gruss P. Pax-6, a murine paired box gene, is expressed in the developing CNS. Development 1991; 113:1435-49.

20. Sisodiya SM, Free SL, Williamson KA, Mitchell TN, Willis C, Stevens JM, Kendall BE, Shorvon SD, Hanson IM, Moore AT, van Heyningen V. PAX6 haploinsufficiency causes cerebral malformation and olfactory dysfunction in humans. Nat Genet 2001; 28:214-6.

21. Free SL, Mitchell TN, Williamson KA, Churchill AJ, Shorvon SD, Moore AT, van Heyningen V, Sisodiya SM. Quantitative MR image analysis in subjects with defects in the PAX6 gene. Neuroimage 2003; 20:2281-90.

22. Mitchell TN, Free SL, Williamson KA, Stevens JM, Churchill AJ, Hanson IM, Shorvon SD, Moore AT, van Heyningen V, Sisodiya SM. Polymicrogyria and absence of pineal gland due to PAX6 mutation. Ann Neurol 2003; 53:658-63.

23. Aguiar PH, Liu CW, Leitao H, Issa F, Lepski G, Figueiredo EG, Gomes-Pinto F, Marino Junior R. MR and CT imaging in the Dyke-Davidoff-Masson syndrome. Report of three cases and contribution to pathogenesis and differential diagnosis. Arq Neuropsiquiatr 1998; 56:803-7.

24. Hentze MW, Kulozik AE. A perfect message: RNA surveillance and nonsense-mediated decay. Cell 1999; 96:307-10.

25. Singh S, Tang HK, Lee JY, Saunders GF. Truncation mutations in the transactivation region of PAX6 result in dominant-negative mutants. J Biol Chem 1998; 273:21531-41.

26. Duncan MK, Cvekl A, Li X, Piatigorsky J. Truncated forms of Pax-6 disrupt lens morphology in transgenic mice. Invest Ophthalmol Vis Sci 2000; 41:464-73.

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