Molecular Vision 2007; 13:1555-1561 <>
Received 18 April 2007 | Accepted 27 August 2007 | Published 30 August 2007

Two novel PAX6 mutations identified in northeastern Chinese patients with aniridia

Huiping Yuan, Yang Kang, Zhenqbo Shao, Yuanyuan Li, Guoyu Yang, Na Xu

Department of Ophthalmology, Harbin Medical University The 2nd Affiliated Hospital, 246 Xuefu Road, Harbin, Heilongjiang 150086

Correspondence to: Dr. Huiping Yuan, Department of Ophthalmology, Harbin Medical University The 2nd Affiliated Hospital, 246 Xuefu Road, Harbin, Heilongjiang 150086; Phone: 86-451-55969288; FAX: 86-451-86605757; email:


Purpose: The PAX6 gene encodes a transcriptional regulator involved in oculogenesis and other developmental processes such as aniridia, a congenital condition characterized by the underdevelopment of the eye's iris. Aniridia may be broadly divided into hereditary and sporadic forms. The function of the PAX6 gene in these two forms of aniridia is still poorly defined. Therefore, we carried out a mutation analysis of the PAX6 gene in northeastern Chinese families with aniridia to identify the role of the PAX6 gene in hereditary aniridia.

Methods: Five aniridia patients from two northeastern Chinese families (Family 1 and Family 2) underwent full ophthalmologic examinations. Genomic DNA was prepared from venous leukocytes from these five patients, 10 non-carriers in these two families, as well as 100 healthy normal controls. The coding regions of PAX6 were analyzed by PCR amplification, direct sequencing and allele-specific cloning sequencing.

Results: We identified two novel PAX6 mutations. The first is a 9 base pair (bp) deletion in exon 5 (c.483del9) that makes a PAX6 protein with de novo in-frame deletions of aspartic acid, isoleucine, and serine at the amino acid codon positions 41-43. The second is a heterozygous mutation (IVS10+1G>A) located at the boundary of exon 10 and intron 10.

Conclusions: We identified two novel PAX6 mutations in familial aniridia from northeastern China, an ethnic group that is not well-studied. The genetic analysis confirms that these two novel mutations in PAX6 are capable of causing the classic aniridia phenotype. Therefore, by studying human familial aniridia cases, we demonstrated that PAX6 plays a role in hereditary aniridia.


Aniridia is a bilateral and panocular disorder with an incidence of 1:64,000-1:96,000 [1]. In addition to the absence of the iris, in most cases there are also defects of the cornea (opacity), lens (dislocation and/or cataracts), retina (foveal dysplasia), optic nerve (hypoplasia), and the anterior chamber angle (glaucoma) [1]. Aniridia is an autosomal dominant inheritance disease with approximately two-thirds of all cases being familial. Autosomal dominant inheritance with almost complete penetrance has also been demonstrated [2]. The remaining one-third is sporadic with no previous family history. However, some of these cases manifest into autosomal dominant inheritance in subsequent generations. A proportion of aniridia is associated with the WAGR (Wilms tumor, aniridia, genitourinary disorders, and mental retardation) syndrome [1].

The PAX6 gene (OMIM 607108) was first identified as a member of the vertebrate PAX family due to its homology to Drosophila paired box genes [3] and was then suggested by positional cloning to be a candidate gene underlying abnormal eye development found in diseases such as aniridia [2]. PAX6 is located on chromosome 11p13 and encodes 422 amino acids as a transcriptional regulator, occupying 14 exons in a 22 kilobase (kb) genomic region (Figure 1). It has two DNA-binding domains, a bipartite paired domain (PD) and a paired-type homeodomain (HD), and a C-terminal proline, serine, and threonine-rich trans-regulatory domain (PST). The PD and HD, which are separated by a linker region (LNK), are the structural basis for the binding activity of the PAX6 protein. The PST domain is thought to activate the expression of downstream genes in cells [4,5]. There are two major isoforms of PAX6: PAX6(-5a), comprised of 422 amino acids, and PAX6(+5a), comprised of 436 amino acids resulting from the insertion of a 14 amino acid-long sequence in the PD. This insertion abolishes PAX6 DNA-binding properties and when overexpressed, induces a developmental cascade modifying the neuronal architecture of the retina [6]. There is a high degree of homology in the coding region. For example, the human and rodent proteins are 100% identical, with chick (96%) and zebrafish (93%) also showing strong homology. The PD and HD of mammals maintain 80-90% similarity to Drosophila and C.elegans [7], suggesting that the DNA-binding targets may also be highly conserved across evolution.

The role of PAX6 in human eye disease has been confirmed through the identification of a large number of mutations. Considerable insight into PAX6 function has been gained from detailed genotype-phenotype comparisons. The features of aniridia reflect the wide expression of PAX6 in the developing eye in the neurectoderm, the surface ectoderm, and their derivatives [8]. Expression continues in the adult retina, lens, and cornea [9,10]. The fact that aniridia is a progressive condition may reflect the maintenance functions of PAX6 in the adult eye. To date, nearly 300 PAX6 mutations have been identified in aniridic subjects worldwide [11]. It is widely recognized that over 75% of all aniridia cases are caused by mutations that introduce a premature termination codon into the PAX6 open reading frame. In contrast, most non-aniridia phenotypes are associated with missense mutations. In addition, studies show that nonsense-mediated decay is a major mechanism acting on PAX6 mutant alleles. In-frame-shifting insertions or deletions are uncommon types of lesions accounting for 5.6% of all aniridia-associated mutations identified in PAX6.



The two Chinese families with aniridia in this study were from Heilongjiang province in northeastern China. Five aniridia patients from these two unrelated families, their 10 non-carrier relatives, and 100 healthy normal controls were recruited for this study. After obtaining the tenets of the Declaration of Helsinki approval and informed consent from patients and their parents, medical and ophthalmic histories were obtained. A glaucoma specialist evaluated the patients and relatives, and individuals with congenital anomalies other than aniridia (for instance, Axenfeld-Rieger syndrome, iridocorneal endothelial syndromes, sclerocornea, or Peter's anomaly) were excluded after a careful clinical ocular evaluation. All patients underwent full ophthalmologic examination including slit lamp biomicroscopy, measurement of intraocular pressure (IOP) by applanation tonometry, gonioscopic evaluation of the anterior chamber angle, and perimetry by automated field analyzer, where appropriate. Systemic evaluation was performed to exclude associated anomalies like Wilms' tumor, urogenital anomalies, and mental retardation in all subjects included in this study.

Molecular methods

Blood samples were drawn by venipuncture and genomic DNA was extracted using the QIAmp Blood kit (Qiagen,Germany). Eight new pairs of primers (Table 1) were used to amplify the 11 coding exons (exon 4 to exon 13 and an extra exon, 5a) and the adjacent intronic sequence of PAX6 (GenBank NC_000011 for gDNA, NM_001604 for cDNA, and NP_001595 for protein). The DNA was subsequently purified using a Qiaex II kit (Qiagen, Hilden, Germany) and sequenced with the ABI BigDye Terminator Cycle Sequencing kit v3.1, (ABI Applied Biosystems, Foster City, CA). The superimposed mutant PCR products were subcloned into pGEM-T vector (Promega, Madison, WI) and sequenced to identify the mutation. Mutation descriptions follow the nomenclature recommended by the Human Genomic Variation Society (HGVS).

Any detected variation in PAX6 was further evaluated in ten family members as well as in 100 normal controls by using heteroduplex PCR-SSCP analysis as previously described in the literature [12]. Two additional pairs of primers for heteroduplex PCR-SSCP analysis [13] were synthesized for the amplification of exon 5 (2) and exon 10 (2) and the detail sequence of primers are listed in Table 2.


For Family 1, direct sequencing (Figure 2B) of PAX6 in three patients (II:1, III:1, and III:2) showed a pattern of peak overlap characteristic of superimposed sequences due to heterozygosity for insertion/deletion events. Allele-specific sequencing (Figure 2C) of the patients' PAX6 subcloned products demonstrated short intragenic deletions in all subjects. This result indicates that there is a 9 bp deletion in exon 5 (c.483del9) that makes a PAX6 protein with de novo in-frame deletions of aspartic acid, isoleucine, and serine at amino acid codon positions 41, 42, and 43.

Family 2 exhibited a heterozygous mutation (Figure 3B) only in intron 10 (IVS10+1G>A) adjacent to exon 10 but not in any other coding exon. Mutations were confirmed by sequencing both sense and antisense strands in the patients and in the unaffected families. The two mutations were also detected by single strand conformational polymorphism (SSCP) analysis (Figure 4A,B) but none were detected in other relatives or control samples (Figure 4C,D). These two mutations have not been previously reported according to the Human PAX6 Allelic Variant Database.

Clinical findings

In Family 1, four individuals in three successive generations were found to have similar congenital ocular disease (Figure 2A). The three living patients were a 38-year-old father (patient II:1) and two daughters, age 16 (patient III:1) and age 15 (patient III:2). Their similar phenotype is characterized by bilateral aniridia, clear and normal sized corneas, and jerk horizontal nystagmus. They suffered from subnormal vision from early childhood, probably from birth. For all three subjects, applanation tonometry revealed normal intraocular pressure in both eyes. Patient II:1 suffered from complete bilateral defects of the iris and a congenital punctate cataract leading to a visual acuity of 20/200 in the right eye (RE) and 20/200 in the left eye (LE). Patient III:1 had only a partial iris defect and a congenital punctuate cataract, and her visual acuity was 40/200 (RE) and 20/200 (LE). Congenital lapsus palpebrae superioris, congenital punctuate cataracts, and features of foveal hypoplasia with absent foveolar and perifoveal reflexes were observed in patient III:2 and her visual acuity was 20/200 (RE) and 10/200 (LE). There were no other abnormalities found in the retina, choroids, or optic nerve head in the family.

In Family 2, the defining characteristic (Figure 3A) was early stage secondary glaucoma due to dysplasia of the trabecular reticulum in the anterior chamber angle. The 39-year-old mother (patient II:4) underwent trabeculectomy for both eyes at the age of 24 upon the onset of acute glaucoma. However, the surgery did not prevent increasing intraocular pressure or decreasing visual acuity. She could detect light perception in her LE (Figure 5A) and but not in her RE (Figure 5B) when this study was performed. Other clinical symptoms included atrophy of the eyeball and corneal leucoma in the RE and slight eyeball horizontal tremors, iris hypoplasia, and lens opacity in both eyes. Her nine-year-old daughter (III:2; Figure 5C) exhibited total bilateral aniridia, slight oscillatory nystagmus, new vessels around the cornea, congenital coronary cataracts, and foveal hypoplasia (Figure 5D). At her first evalution, she demonstrated a visual acuity of 50/200 (RE) and 40/200 (LE), an intraocular pressure of 28 mmHg, and incipient glaucomatous changes in the optic cup. Subsequently, she began systemic treatment for glaucoma.


Aniridia is a human eye malformation caused by heterozygous null mutations of PAX6, a paired box transcription factor, or by microdeletions of chromosome 11p13 that encompasses PAX6 and is associated with the WAGR syndrome. PAX6 is extraordinarily conserved throughout evolution with the murine and human proteins differing by only one amino acid over their entire length. Moreover, zebrafish PAX6 is 97% identical to murine and human PAX6 at the amino acid level. Such a high degree of conservation argues for conserved functional importance of virtually every amino acid residue. Entire deletions of PAX6 (e.g., WAGR patients) and intragenic PAX6 null mutations manifest no clear phenotypic differences. The molecular basis for aniridia are derived from mutations of PAX6 that lead to premature protein termination, which is assumed to cause loss of function of one allele, resulting in 50% reduction in overall activity, rather than from the accumulation of dominant or dominant-negative forms of the protein. Therefore, haplo-insufficiency of PAX6 has been suggested to underlie the aniridia phenotype.

In the present study, we described two novel mutations (c.857del9 and IVS10+1G>A) in PAX6 in two northeastern Chinese families with aniridia. In-frame insertions or deletions in PAX6 account for only 5.6% of all gene lesions described so far, making it the second most uncommon mutation (run-on mutations account for 4.8%) [11]. According to the Human PAX6 Allelic Variant Database, all the in-frame deletions of PAX6 arise in a highly conserved region among paired domain corresponding to the helix-turn-helix (HTH) configuration [14] and it is predicted to alter the ability of the PAX6 protein to bind its target DNA sequences. In Family 1, the dysfunction of PAX6 is likely due to a de novo in-frame deletion of three amino acids (D41, I42, and S43) in the PD. Codons R38 and P39, are conserved in all PAX family members [14]. Crystal structure analysis of the human PAX6 PD [15] demonstrates that codons D41, I42, and S43 lie on the second alpha helix of the HTH motif. There are three contiguous invariant amino acids (R38, P39, and C40) adjacent to these codons that are in contact with the phosphate group of the sugar-phosphate backbone of the target DNA. Therefore, it is likely that the in-frame deletion of these codons in these patients not only disrupts the DNA backbone contact site but also interferes with the correct folding of the HTH configuration in the PD. The latter might change the three-dimensional structure of the PD, particularly the N-terminal paired subdomain that is thought to cause loss of function of one allele. This would result in a 50% reduction in overall activity which is sufficient to cause the aniridia phenotype.

Three affected members from Family 1 had different phenotypes but the same mutation of PAX6. Although different phenotypes caused by the same PAX6 mutation have been reported previously [16], this makes the analysis of genotype-phenotype correlation more difficult. However, missense mutations in exons 4-6, which involve the PD, may cause less severe and more variant phenotypes [17]. Some hypomorphic alleles with reduced activity may contribute to less severe or variant phenotypes [18,19]. In that case, we can deem that in-frame deletions in the PD would cause similar phenotypes. Variant phenotypes are mostly related to missense mutations, indicating that altered function of the protein may trigger distinct or milder phenotypes when compared with complete loss of function. Correlating phenotype to the site of the nonsense mutation has been unsuccessful as the truncated protein may not be produced due to nonsense-mediated decay of the mRNA [20]. We speculate that there is a milder effect of a mutation that preserves the PD and HD binding properties.

Family 2 carried a heterozygous (IVS10+1G>A) mutation in position +1 of the exon 10-intron 10 boundary. This mutation resulted in aberrant mRNA splicing and in skipping of exon 10. The splicing site mutations resulted in loss of the corresponding splicing site, causing a deletion of 151 bp belonging to exon 10, which instigated premature termination of the PAX6 protein. Loss of one functional allele results in 50% reduction of PAX6 function (haplo-insufficiency), which has been suggested to contribute to the ocular defects seen in aniridia [11,20]. A noticeable characteristic of the family is the onset of secondary glaucoma at the early stage that was probably caused by dysplasia of the trabecular reticulum. Future studies should examine whether there is a correlation between the site effect of PST domain and dysplasia of the trabecular reticulum in the anterior chamber angle.

In summary, our data add two novel mutations found in the northeastern Chinese population to the existing spectrum of PAX6 mutations. These findings provide further examples of haplo-insufficiency of PAX6 in aniridia.


The authors are grateful to all patients, their families, and normal volunteers for their participation in this investigation. This study was supported by prominent youth Science Foundation of Heilongjiang province (JC200620).


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