2.1.Subjects:The proband and the proband’s family 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(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.
2.2.Clinical Evaluation: Thorough ophthalmologic examination were performed to the proband and her brother, including tests of visual acuity, intraocular pressure(IOP),slit-lamp analyses, anterior segment photography, visual ﬁeld tests(Humphrey 750,Carl Zeiss, Germany), funduscopy, ultrasonic B analyses(Chiescan Quantel Medical, France), gonioscopic analyses, OCTA(optical coherence tomography angiography)assessments(RTVue-XR Avanti,v2017.1.0;OptoVue,Inc.,CA,USA),and ultrasound biomicroscope(UBM)assessments(SW China),the proband’s other family members underwent a simple slit lamp examination.
2.3.1.Genome DNA extraction:About 4 ml of venous blood was sampled from proband and proband’s brother. The genomic DNA samples were stored at -20℃ before using.
1.Genome-wide library construction: DNA enzymatic fragmentation and genome-wide library construction were carried out using the DNA library construction kit of YEASEN Biology Company（Hieff NGS® OnePot DNA Library Prep Kit for Illumina®，YEASEN).
2.Construction of clinical whole exon group capture library:
XGen Exome Research Panel V1.0 （Integrated DNA Technologies, Inc.，USA）of IDT company was used to capture the clinical all exon group library and constructed the clinical all exon group library of the proband and his brother.
2.3.3.Clinical total exome sequencing：Two-terminal sequencing was performed on the Illumina (san diego, ca) sequencing platform using PE 150 patterns.
Primer3Plus（http://www.primer3plus.com/cgi-bin/dev/primer3plus.cgi）was used to design primers for PAX6 gene C. 114_119delinsAATTTCC(P :Pro39fs) site and in-silico PCR（http://genome.ucsc.edu/cgi-bin/hgPcr）was used to verify the specificity of the primer(Table 1). PCR amplification products of family members were sequenced using ABI3730s AUTOMATIC DNA sequence Analyzer (3730 DNA Analyzer), and sequencing results were analyzed and compared using CodonCode Aligner software (CodonCode Corporation, USA).
Table 1.Sequencing primer details.
2.3.5. Analysis: Raw reads of low quality were removed, and the remaining reads were mapped to the UCSC(University of California Santa Cruz) hg19 reference genome(http://genome.ucsc.edu/). Single nucleotide variations(SNVs)and insertion-deletion(InDel)mutations were detected using the HaplotypeCaller function of the Genome Analysis ToolKit(GATK,http://software broadinstitute org/gatk/).These annotated variants were then filtered based on the Annovar(http://www.openbioinformatics.org/annovar/)database,The databases used in mutative pathogenicity prediction included SIFT(http://sift.jcvi.org), Polyphen2_HDIV(http://genetics.bwh.harvard.edu/pph2), Polyphen2_HVAR(http://genetics.bwh.harvard.edu/pph2), LRT(http://www.genetics.wustl.edu/jflab/lrt_query.html),MutationTaster(http://www.mutationtaster.org/), MutationAssessor(http://mutationassessor.org/r3/), FATHMM(http://fathmm.biocompute.org.uk), PROVEAN(http://provean.jcvi.org/index.php), MetaSVM(https://omictools.com/meta-svmtool), MetaLR(http://www.ensembl.info/tag/metalr/), M-CAP(http://bejerano.stanford.edu/mcap/), fathmm-MKL_coding(http://fathmm.biocompute.org.uk/fathmmMKL.htm). Quality control requirements:data volume>=6GB,average coverage>=150X,30X coverage>=98.5%,Q30 Qualification rate (%)>=89.17%).
2.3.6.Interpretation:The guidelines of the American College of Medical Genetics and Genomics(ACMG)were used to facilitate appropriate data analysis（Table 3）. Only those genetic variations with known, definitive genetic associations were analyzed. 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.
Table 3.Classification basis referred to ACMG guide.