Detection of a novel PAX6 mutation in a Chinese family with multiple ocular abnormalities

Background: Aniridia is a congenital,panocular disease affecting the cornea,anterior chamber angle,iris,lens,retina and optic nerve.PAX6 loss-of-function mutations were the most common cause of aniridia.Mutations throughout the PAX6 gene have been linked to a range of ophthalmic abnormalities,with distinct mutations at a given site within this gene leading to distinct phenotypic ndings.This study aimed to characterize genetic mutations associated with congenital aniridia in a Chinese family. Methods:The proband and the proband’s brother of this family underwent comprehensive ophthalmologic examinations as well as exome sequencing,with Next Generation Sequencing being used to conrm these results. Results: A novel mutation(c.114_119delinsAATTTCC:p.Pro39fs)in the PAX6 gene was identied in subjects III-2 and III-3 in these family,and both of these subjects exhibited complete aniridia,cataracts,glaucoma,high myopia,and foveal hypoplasia. Conclusions: We identied a novel PAX6 frameshift heterozygous deletion mutation in a Chinese family and determined that this mutation was a probable cause of various eye abnormalities in carriers. ,although


Background
Aniridia is a condition that is de ned by a near or total absence of an iris,and can be either acquired or congenital.Congenital aniridia is a rare condition that affects 1:64,000-1:96,000 people and is often sporadic [1] ,although up to two-thirds of patients exhibit an autosomal dominant form of aniridia [2].
While the lack of an iris is the most salient hallmark of this condition,congenital aniridia affects both eyes and is also associated with abnormalities in the cornea,the retina,the lens,the anterior chamber angle,and the optic nerve.Most aniridia patients exhibit macular hypoplasia,nystagmus,and signi cant visual impairment,with a smaller subset of patients also suffering from optic nerve hypoplasia [3].Patients with aniridia also often suffer from a range of secondary ocular complications including cataracts,aniridic keratopathy,and glaucoma,with the latter of these conditions affecting up to 70%of aniridic patients [4] .
To date,over 500 mutations in the PAX6 gene and its regulatory regions have been characterized,with many of these mutations resulting in PAX6 haploinsufficiency that can cause signi cant ocular and systemic abnormalities [5] .To date,however,few studies have characterized hybrid frameshift deletion insertion PAX6 mutations associated with ocular diseases in Chinese families.In the present report,we therefore describe a novel PAX6 mutation that was found to be associated with congenital aniridia in a Chinese family. Methods 2.1.Subjects:The proband and the proband's brother with a history of congenital aniridia were recruited at the Aier Eye Hospital of Changsha,the father of the proband had also been diagnosed with aniridia at a different hospital,Other members of the family had no eye symptoms( Figure 1).This study was approved by the Aier Eye Hospital of Changsha ethics committee and was consistent with the Declaration of Helsinki,with all subjects providing informed consent to participate.

2.3.Mutation screening
2.3.1.Sequencing:About 4 ml of venous blood was sampled from proband and proband's brother.Sequences samples were prepared based on appropriate protocols for the Sure Select Target Enrichment System Capture Process(Illumina HiSeq X),after which exome sequencing was conducted.

2.3.3.Interpretation:
The guidelines of the American College of Medical Genetics and Genomics(ACMG)were used to facilitate appropriate data analysis.Brie y,only those genetic variations with known,de nitive genetic associations were analyzed,and genes with unknown pathogenicity or functionality were omitted from these analyses.In addition,common benign polymorphic variants,synonymous variants,and intronic variants not altering mRNA splicing were not included in these analyses unless they have previously been reported in the literature as being pathogenic or were included in the database.

Clinical data
Proband A 13-year old girl presented to our hospital complaining of bilateral blurred vision,with no history of surgery or medical treatment of either eye.Goldmann tonometry revealed her IOP to be 44 mmHg OD and 38mmHg OS,with her best-corrected visual acuity(BCVA)being 20/100 OD and 20/125 OS.Further evaluation revealed refractive errors of-8.5 D OD and-6.0 D OS,with eyeball axis length(AL)values of 26.3 mm OD and 26.0 mm OS.The anterior chamber in both eyes appeared normal,with peripheral angles being open in both eyes.A slit lamp revealed the presence of bilateral peripheral cataracts and posterior capsular opaci cation,while UBM examination revealed iris coloboma.A further fundus examination a large optic disc with bilateral glaucomatous cupping and peripapillary atrophy,while OCTA examination revealed diffuse superior and inferior RNFL(retinal nerve bre layer)thinning,reduced wiVD(whole image vessel density),idVD(inside disc vessel density),and ppVD(peripapillary vessel density)vessel density,and signi cant foveal hypoplasia.Visualfield tests highlighted bilateral glaucomatous visual eld defects(Table 1, Figure 2).The proband had no other discomfort, especially hearing loss or abnormal olfaction.

Brother of the Proband
The 23-year-old brother of the proband reported a history of glaucoma that had been diagnosed at a different hospital one year prior,and was managing this condition with IOP-lowering eye drops.With glasses,his BCVA in both eyes was 20/80,with refractive error values of-9.5 D OD and-10.25 D OS,and with eyeball AL values of 26.7 mm OD and 26.5 mm OS.He exhibited many of the same ophthalmic abnormalities as did his sister,including complete aniridia,cataracts,glaucoma,high myopia,and foveal hypoplasia.In addition,the brother exhibited a decreased VD relative to that normally observed in healthy eyes.The superior and inferior RNFL of the brother's eyes were thicker relative to his sister's eyes,and he exhibited less pronounced bilateral glaucomatous visual eld defects(Table 1, Figure 3). Proband's brother had no hearing loss or abnormal olfaction either. .

Mutation analysis
Second-generation sequencing analyses revealed the presence of a heterozygous frameshift deletion mutation(c.114_119delinsaatttcc:p.P ro39fs)in exon 5 of the PAX6 gene.This mutation,which consisted of a 6 bp deletion and a 7 bp insertion,resulted in a frameshift in the PAX6 gene from the 39th proline codon resulting in the generation of a premature stop codon( Figure 4).According to the ACMG guidelines, the mutation is a possible pathogenic variant ( Table 2).

Discussion
By analyzing a Chinese family with a history of congenital aniridia,we herein identi ed a novel hybrid mutation(C.114_119delinsAATTTCC:p.Pro39fs)in the PAX6 gene.This mutation consisted of a 6 bp deletion and a 7 bp insertion that resulted in the premature truncation of the PAX6 protein.The affected brother and sister patients exhibited shared ophthalmic abnormalities including cataracts,nystagmus,glaucoma,aniridia,and macular fovea hypoplasia.The PAX6 gene was rst characterized by Ton et al.in 1991 [6] ,and is found on chromosome 11p13.PAX6 encodes a transcriptional regulator that is important for the development of organs and tissues including the eyes.PAX6 expression is detectable in the iris,lens,optic disc,corneal epithelium,ciliary body,retinal neuroepithelium,and retinal pigment epithelium.In 2005,Tzoulaki et al.characterized human PAX6 mutations and found that mutations throughout this gene were associated with aniridia and related phenotypes [7] .In an additional study of 95 Chinese patients with aniridia,You et al.found that PAX6 loss-of-function mutations were the most common cause of aniridia [8] .The identi ed PAX6 mutation in these siblings(c.114_119delinsAATTTCC:p.Pro39fs)resulted in a frameshift from the 39th codon of this gene,resulting in the premature generation of a stop codon.In light of prior studies,we hypothesized that this mutation was likely to be the primary cause of aniridia and other observed ophthalmic abnormalities in these siblings.
In their prior study of 95 Chinese aniridia patients,You et al.identi ed 47 different mutations associated with the aniridia phenotype including 6 frameshift InDel mutations,12 nonsense mutations,2 missense mutations,1 run-on mutation,1 synonymous mutation,and 15 mutations that altered mRNA splicing [8] .The human gene mutation database(HGMD)currently includes 479 pathogenic PAX6 mutations gene(http://www.hgmd.cf.ac.uk/ac/gene.php?gene=PAX6).In total,20 reports to date have described cases of patients with both insertion and deletion mutations in the PAX6,and such combination mutations are generally more likely to be associated with serious ophthalmic abnormalities.Our observations of abnormalities including aniridia and glaucoma in the patients in the present study are thus consistent with these prior studies.
The PAX6 protein is composed of four domains:two DNA-binding domains,including an N-terminal 128 amino acid paired box domain(PD)and a 61 amino acid homeodomain(HD),as well as a 79 amino acid glycine-rich hinge region and a C-terminal proline-rich serine transactivation domain [9,10] .Mutations throughout the PAX6 gene have been linked to a range of ophthalmic abnormalities,with distinct mutations at a given site within this gene leading to distinct phenotypic ndings.Glaucoma manifested at an earlier age and was more severe in the proband than in her brother in the present study.Two primary models have been proposed to describe the penetrance of PAX6 mutations.Dominant-negative PAX6 mutations are thought to enhance PAX6 binding to DNA,leading to abnormal dominant-negative effects as a result of premature PAX6 truncation [11] .Other PAX6 mutations are better described by a dose-effect model wherein premature termination codons(PTCs)within the PAX6 open reading frame lead to premature protein truncation as a result of nonsense-mediated mRNA decay(NMD).In such a dose-effect model,a single wild-type allele of PAX6 is insu cient to facilitate normal ocular development,leading to the observed ophthalmic abnormalities [12] .Subtle phenotypic differences between patients with different PAX6 mutations may thus be attributable to slight differences in intracellular PAX6 levels [13] .In the present study,we identi ed a novel heterozygous frameshift mutation in PAX6 that resulted in a frameshift from the 39 th proline codon and the generation of a premature stop codon.This mutation began in exon 5 in the PD domain and led to the truncation of the functional [14] .Haploinsu ciency is likely to explain the observed aniridia phenotype in the subjects of the present study,although the mechanistic link between genotype and phenotype in these patients remains to be fully characterized in future studies. This study has multiple limitations.For one,per the ACMG guidelines,the identi ed mutation can at present only be identi ed as a possible pathogenic or clinically unknown mutation that may be linked to phenotypic ndings in study subjects.In addition,we identi ed other mutations that were considered to be beyond the scope of the present study.This study was also unable to identify certain structural mutations(including inversions,rearrangements,and ectopic mutations),dynamic mutations,low prevalence chimeric mutations,regulatory region mutations,or epigenetic variability.Furthermore,owing to the existence of highly repetitive low-complexity regions and homologous sequences,certain exonic regions have little to no coverage in our analyses.

Conclusions
In summary,we identi ed the novel heterozygous c.114_119delinsAATTTCC:p.Pro39fs mutation the PAX6 gene as a putative cause of aniridia in a Chinese family.These results expand the spectrum of known mutations that can cause PAX6-triggered congenital aniridia,while also enhancing current understanding regarding the genetic etiology of this condition.Our ndings further have the potential to aid in the genetic diagnosis of aniridia.

Declarations
Ethics approval and consent to participate:Approved by Changsha Aier Eye Ethics Committee (2019)KYPJ001.Written informed consent was obtained from the proband,the proband's brother and their father. Consent for publication:We con rm that the manuscript has been read and approved by all named authors and that there are no other persons who satis ed the criteria for authorship but are not listed.We further con rm that the order of authors listed in the manuscript has been approved by all of us.The brother and the father of the the proband that was a minor gave written consent for their personal or clinical details along with any identifying images to be published in this study.
Availability of data and materials:All data generated or analysed during this study are included in this published article[and its supplementary information les].
Competing Interests:The authors declare that they have no competing interests.  Table   Table 1.RNFL,wiVD(whole image vessel density),idVD(inside disc vessel density),and ppVD(peripapillary vessel density)values for the proband and the proband's brother.  Table 2 ACMG Description of evidence Classification results

PVS1
The pax6 gene in the clingen database(https://clinicalgenome.org/) was recorded with a single dose sensitive gene with a score of 3 . Possible pathogenicity

PM2
It is a rare variant, not included in the Genome Aggregation Database (gnomAD,https://gnomad.broadinstitute.org/about) East Asian database. Figure 1 Pedigree of a Chinese family with aniridia.Squares and circles correspond to males and females,respectively.Black and white shapes correspond to affected and unaffected individuals,respectively.The proband is indicated with an arrow.

Figure 2
Clinical ndings in the proband.The right and left columns correspond to the left and right eyes,respectively.Images of the anterior segment(a,b)and horizontally scanned UBM images of the anterior chamber(c,d).Images of the optic disc(e,f),images of B-mode ultrasound(g,h)and pattern deviation plots from the Humphrey750 visual eld test(i,j).Vertically scanned ultrasonic B images of the eyeball.

Figure 3
Clinical ndings in the proband's brother.The right and left columns correspond to the left and right eyes,respectively.Images of the anterior segment(a,b)and horizontally scanned UBM images of the anterior chamber(c,d).Images of the optic disc(e,f),images of B-mode ultrasound(g,h)and pattern deviation plots from the Humphrey750 visual eld test(i,j).Vertically scanned ultrasonic B images of the eyeball.

Figure 4
Next Generation Sequencing revealed the presence of a frameshift heterozygous deletion mutation(red box)in the proband(a)and the proband's brother(b).Sanger Sequencing con rmed the mutation sites(red arrow),with the dual peaks corresponding to the presence of the indicated heterozygous insertion variants in the proband(c)and the proband's brother(d).