Molecular analysis of Cypriot families with aniridia reveals a novel PAX6 mutation.

The present study investigated the clinical and mutational spectrum of aniridia in a cohort of 17 affected individuals from six families from Cyprus. Each proband was initially evaluated for copy number variants at the PAX6 locus and subsequently underwent PAX6 mutation screening. Sequence analysis of FOXC1 and PITX2 was performed in patients who did not carry a PAX6 mutation. The most common clinical features in the group of aniridia patients associated with aniridia were nystagmus, cataracts and glaucoma. PAX6 pathogenic mutations were identified in five out of six families (a diagnostic yield of 84%). Previously reported pathogenic mutations in PAX6 were identified in four families, which comprise p.R203*, p.R240* and p.R317*. In addition, a novel pathogenic variant (p.E220Gfs*23) was identified in a single family. No pathogenic mutations were detected in PAX6, FOXC1 or PITX2 in the only patient with a sporadic form of aniridia‑like phenotype, confirming the genetic heterogeneity associated with this disease. To the best of our knowledge this is the first report on the mutational spectrum of PAX6 in aniridia patients of Cypriot ancestry. Mutational screening of PAX6 serves a crucial role in distinguishing isolated from syndromic forms of aniridia, and it may therefore eliminate the need for renal ultrasound scan surveillance, delineate the phenotype and improve genetic counseling.

Introduction

Aniridia (MIM #106210) is a congenital disorder of complete or partial iris hypoplasia (1,2). The prevalence of aniridia ranges from 1:50,000 to 1:100,000 live births (3). Aniridia can occur as an isolated or a syndromic form (4). Approximately two thirds of all cases are familial following an autosomal dominant mode of inheritance with high penetrance, while the remaining cases are sporadic (3,5).

Classic aniridia is a panocular condition caused by PAX6 heterozygous mutations and it affects the iris, cornea, lens, retina and optic nerve. It can be accompanied by foveal hypoplasia, strabismus and optic nerve hypoplasia, generally leading to impaired visual acuity, while late-onset manifestations can include nystagmus, glaucoma, cataract and corneal pannus (5,6). Patients may also display non-ocular sensory and neurological abnormalities, such as reduced olfaction and hearing difficulties (7), and a range of neuroanatomical abnormalities, including hypoplasia of the anterior commissure, the pineal glands and the optic chiasm (8,9). PAX6 point mutations are responsible for classic aniridia in approximately 90% of patients (10,11). PAX6 maps to chromosomal region 11p13 and encodes a transcription factor, which plays a crucial role in early ocular morphogenesis. It is also involved in the development of the central nervous system, gut and pancreas (12,13). Furthermore, deletions at 11p13 involving PAX6 or the regulatory region upstream of PAX6 leaving its coding region intact are thought to be rare causes of classic aniridia (14). A small number of patients develop aniridia as part of the WAGR syndrome (Wilms tumor, Aniridia, Genital anomalies, mental Retardation) caused by a contiguous gene deletion encompassing PAX6 and WT1 (4). Finally, about 10% of cases display aniridia-like phenotypes that result from mutations in other genes, such as FOXC1 and PITX2 (15–17). The purpose of this study was to analyze the PAX6 gene in a group of aniridia patients of Cypriot ancestry and describe their clinical features. We identified three previously reported PAX6 mutations, in addition to a novel frameshift variant that was identified in one of our familial cases.

Materials and methods

Patients

A total of 17 affected individuals from six families were evaluated with a complete ophthalmological examination and then referred to the Clinical Genetics Clinic at the Cyprus Institute of Neurology and Genetics to investigate for PAX6 mutations. Informed consent was obtained by study participants or their guardians if they were younger than 18 year olds.

Array-comparative genomic hybridization (CGH)

Array CGH analysis or multiplex ligation-dependent probe amplification assay (MLPA) were initially performed to investigate for whole gene deletions involving PAX6 and/or WT1 or deletions downstream of PAX6. Array-CGH was performed using the Cytochip ISCA array (version 1.0; BlueGnome, Cambridge, UK) with 180,000 oligos in a 4×180 k format. Fluorescent ratios were calculated using the Blue Fuse Multi Software (version 4.2; BlueGnome). MLPA was conducted using the SALSA probemix P219-B3 (MRC-Holland, Amsterdam, Netherlands). Fragment separation by capillary electrophoresis was performed on an ABI 3130×l genetic analyzer. MLPA analysis was performed using the Coffalyser.Net Software (version 1.4; MRC-Holland).

Mutation screening

Genomic DNA was isolated from peripheral blood using the QIAamp DNA Blood Midi Kit (Qiagen, Inc., Valencia, CA, USA). All coding exons of PAX6, FOXC1 and PITX2 were amplified by PCR using primers designed with Primer3 (http://frodo.wi.mit.edu/) and they are available upon request. Sequencing products were analyzed by a 3130×l Genetic Analyzer (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The cDNA sequence of the most common human PAX6 transcript (NM_000280) is used for the variant nomenclature. Direct sequencing of PITX2 and FOXC1 was performed in patients who did not carry any PAX6 mutations only.

Results

Seventeen patients from six families were recruited for this study, including eight males and nine females. One patient was a sporadic case while the remaining were familial ones. Patients from family 1 presented with isolated bilateral aniridia. The proband of family 2 presented with bilateral aniridia, cataracts, glaucoma of the left eye, nystagmus and reduced visual acuity. She also had pseudophakia of the left eye. Fundoscopy revealed small, hypoplastic discs, macular hypoplasia, mild foveal hypoplasia and optic nerve hypoplasia. Her mother had aniridia with loss of vision in the right eye and developed retinal detachment in the left. The proband of family 3 had bilateral aniridia, nystagmus, cataracts, glaucoma and photophobia. She also had a history of osteoporosis and hypercholesterolemia. Her mother had relapsing remitting multiple sclerosis in addition to aniridia. The proband of family 4 had bilateral aniridia, atrial septal defect and thyroid nodules. Patients from family 5 exhibited bilateral aniridia and cataracts. Finally, our sporadic patient from family 6 had bilateral aniridia, nystagmus, cataracts and glaucoma. He had a history of Peters anomaly and he showed markedly abnormal anterior segment. Non-ocular abnormalities included antenatal bilateral hydronephrosis, vesicoureteric obstruction, bilateral megaureter, bipolar disorder and hypertension. His parents were thought to be distantly related. All probands were negative for deletions spanning PAX6 and/or WT1, excluding WAGR syndrome.

Sequencing results

Four different heterozygous PAX6 mutations were identified in five out of six families with aniridia, yielding a diagnostic rate of approximately 84%. Three stop-gain mutations identified in four familial cases (p.R203*, p.R240* and p.R317*) have been reported elsewhere, while the frameshift mutation c.659delA (p.E220Gfs*23) is novel and was found to occur de novo in the affected father (Fig. 1). Mutations were located in exons 8, 9 and 11. All mutations are summarized in Table I.

Figure 1.

Pedigrees of the participating families and their corresponding chromatograms showing the identified mutations in PAX6.

Table I.

Summary of PAX6 mutational spectrum in Cypriot families.

Protein change	Times reported in LOVD	Exon	Protein domain	Predicted effect
p.R203*	40	8	Linker domain	NMD
p.E220Gfs*23	0	8	Linker domain	NMD
p.R240*	51	9	Homeodomain	NMD
p.R317*	41	11	PST domain	NMD

LOVD, Leiden Open (Source) Variation Database; PST, proline-serine-threonine rich transactivation; NMD, nonsense-mediated decay.

Direct sequencing of the coding exons and flanking intronic sites of PITX2 and FOXC1 in the proband of family 5 revealed no pathogenic mutations.

Discussion

In this study, we analyzed 17 patients from six different Cypriot families with aniridia and identified five different PAX6 pathogenic variants in five out of six probands, yielding a diagnostic rate of 84% that is comparable to other studies in other populations (18–21). This is the first study on PAX6 molecular analysis in aniridia patients of Cypriot ancestry. Although, deletions spanning the 3′regulatory region of PAX6 or the PAX6 coding region are less common than PAX6 mutations in aniridia patients, we have performed array-CGH or MLPA first as it was recommended by Hingorani et al due to the clinical importance of detecting WT1 deletions, which requires surveillance for Wilms tumor (7). None of our patients carried a deletion and therefore PAX6 mutation screening was subsequently performed.

To date, 472 unique PAX6 variants have been recorded in the Human PAX6 Allelic Variant Database (http://lsdb.hgu.mrc.ac.uk/home.php?select_db=PAX6). Over 50% of these variants comprise frameshift or stop-gain variants resulting in a premature termination codon, which usually leads to the degradation of the truncated mRNA via nonsense-mediated decay. Therefore, these mutations result in 50% reduction in protein levels supporting haploinsufficiency as the main mechanism underlying aniridia (22,23). All of the identified variants in our study, as well, are predicted to lead to nonsense-mediated decay. Three of the identified PAX6 variants in our study account for 16% of patients included in the PAX6 mutation database (http://lsdb.hgu.mrc.ac.uk/home.php?select_db=PAX6). The first identified mutation (p.R203*) is currently reported in 40 patients, the second one (p.R240*) is currently reported in 51 patients and the third one (p.R317*) is currently reported in 41 patients. These are the most recurrent mutations found in PAX6 in aniridia patients to date.

The PAX6 protein consists of a paired domain at the N-terminus, a homeodomain and a proline-serine-threonine rich transactivation (PST) domain at the C-terminus (24–26). In our study, mutations were found in exons encoding the linker domain (p.R203* and p.E220Gfs*23), the homeodomain (p.R240*) and the PST domain (p.R317*). No correlation between the location of the mutation and the associated phenotypes was observed. Even though all patients had truncating mutations, not all patients had cataract, glaucoma, nystagmus or foveal hypoplasia. In addition, the impairment of visual acuity varied between patients carrying the same PAX6 mutation, as previously observed in other studies (27). The commonest additional ocular features seen in our group of patients were nystagmus, cataracts and glaucoma.

However, no copy number variants or mutations were detected in PAX6, FOXC1 or PITX2 in the proband of family 6 with a sporadic form of classical aniridia. Although, classical aniridia is primarily caused by PAX6 mutations, its phenotypic presentation may overlap with aniridia-like phenotypes. The abnormal anterior segment seen in our patient prompted us to directly sequence FOXC1 and PITX2, because mutations in these genes are more commonly associated with anterior segment dysgenesis even though they can also cause isolated aniridia. FOXC1 mutations are more commonly associated with isolated ocular, heart and/or hearing defects and PITX2 are more commonly associated with ocular, dental and umbilical anomalies. However, both genes account for approximately 40% of cases with Axenfeld-Rieger syndrome only (28,29). Therefore, our molecular findings confirm the genetic heterogeneity that underlies aniridia-like phenotypes as other recent reports have suggested (19,30).

In conclusion, a high diagnostic yield (84%) was obtained in this study, which was the first one to be conducted in Cyprus for aniridia patients. We have identified a novel frameshift mutation in one of our families thus expanding the number of PAX6 mutations that cause aniridia. Mutation screening of PAX6 plays a crucial role in determining whether the affected individual has isolated aniridia or WAGR syndrome. The identification of a PAX6 mutation eliminates the need for surveillance by renal ultrasound and improves genetic counseling as well as the accuracy of prognosis and recurrence risk.