A Novel PAX6 Deletion In A Chinese Family With Congenital Aniridia

Backgrounds and AimsAniridia is a clinical rare clinical congenital inherited ophthalmopathy, charactered with iris of poorly developed or being absent and even with other eye diseases like macular and optic nerve hypoplasia, cataracts, glaucoma, nystagmus, amblyopia, strabismus and refractive errors. Studies have shown that PAX6 is closely associated with congenital aniridia. Globally, the incidence of congenital aniridia is 1/64000 ~ 1/96000 without differences in races, regions, ages or genders. Roughly two-thirds of patients have a family history, and about one-third is sporadic. 85% of congenital aniridia patients are autosomal dominant while 13% are companied with WAGR syndrome, and 2% were autosomal recessive.Material and MethodsWe investigated a autosomal dominant aniridia Chinese family of three generations and recorded the detailed family history from our hospital in August 2014. There are nine members in the family, which includes 2 cases of male patients, 3 cases of female patients, and four normal family members. Blood samples of all these nine family members were collected, and at the same time we collected 100 normal healthy check-up persons’ blood samples randomly. All participants underwent the whole body check in detail and comprehensive bilateral ophthalmological examinations, including best-corrected visual acuity(BCVA), dilated fundus examination using a slit-lamp biomicroscope and colour fundus photography, anterior segment image, etc.DNAs in blood samples were extracted by the phenol-chloroform. Primers were designed according to the 14 exons of PAX6 for specific pieces of polymerase chain reaction(PCR) amplification. PCR products were purified by the agarose gel electrophoresis, and then carried on the bidirectional DNA direct sequencing. Sequencing results were analyzed by DNAStar software.Gene JET RNA purification kit for extracting the RNA in the blood lymphocytes was used, and RNAs were reversely transcripted into c DNA. Maxima SYBR Green q PCR detection kits were used for the real-time fluorescent quantitative PCR. Β-actin gene was used as a reference. The result data was read by Mx3000 P q-PCR software.ResultsAniridia in this family is autosomal dominant inheritance and all patients were characterized by ophthalmic diseases not associated with any other systemic diseases. Five patients have similar clinical manifestations, including congenital aniridia, photophobia, pendular nystagmus, of which 3 cases were adults suffering from cataract and 2 cases were children without cataracts.Analyzing sequencing results by DNAStar software, a novel heterozygous PAX6 deletion mutation c.796 del G(p.A266 fs)(Gen Bank ID: KP255960) in exon 10 was exclusively observed in all affected individuals but not in any of the unaffected family members or unrelated controls.Q-PCR showed that PAX6 m RNA level was about 50% lower in patients with aniridia than in unaffected family members, indicating that this mutation caused nonsense-mediated m RNA decay.Conclusions1. In this study, we found a new mutation in the PAX6(c. 796, del G p.A 266 fs) caused frameshift mutation lesding to premature termination codon in the homeo domain encoding sequence, which resulted in the lack of proline-serine-threonine rich domain.2. The R-T PCR showed that transcription of m RNA from patients with mutational PAX6 was nearly 50% less than that of normal person. This research results was consistent with the theory that the PAX6 shortage of single dose is the phenotype basis of congenital aniridia.3. This discovery not only expands the PAX6 mutation spectrum, but also further verifies the important role of PAX6 during the development of the eye. At the same time, this discovery provides references to the molecular biology research of congenital aniridia, genetic counseling and prenatal diagnosis.