The Institutional Review Board (IRB) of Hainan hospital of Chinese PLA General Hospital (Hainan Province, China) approved the present study. All participating family members provided an informed written consent and were endorsed by their respective IRB. The whole procedure of the present study adhered to the tenets of the Declaration of Helsinki.
A small pedigree with aniridia from Hainan province, China, was recruited for the present study. This family included two affected and two unaffected members, which was analyzed and followed up clinically at Hainan Hospital of Chinese PLA General Hospital (Fig. 1A). Comprehensive ophthalmological examinations, including best correct visual acuity (BCVA), applanation tonometry, dilated funduscopy, anterior segment and fundus photography, ultrasound biomicroscopy (UBM) examination, gonioscopy and optical coherence tomography (OCT) of anterior segment and macular were performed on the affected individuals and the unaffected family members. Genomic DNA was collected from peripheral blood lymphocytes of the pedigree members and normal controls using a QIAamp DNA Blood Midi Kit (Qiagen, Hilden, Germany).
Targeted genes enrichment and sequencing
Target enrichment panel of specific hereditary eye based on the next generation sequencing was used to collect the protein-coding region of 371 targeted genes (designed by MyGenostics, Baltimore, MD), which included 37 genes associated with iris diseases. (PAX6, ELP4, FOXD3, PITX3, FOXE3, PITX2, ADAMTS10, FBN1, LTBP2, ADAMTS17, MTHFR, TYR, MITF, PAX3, SNAI2, SOX10, RBP4, CHD7, TMEM67, RPGRIP1L, CC2D2A, YAP1, MAF, C12orf57, FOXC1, B3GLCT, NF1, GPR143, OCA2, TYRP1, CRYGC, ADAMTSL4, SALL2, IGBP1, CYP1B1, MYOC, SALL1). The genes related to aniridia, WAGR syndrome, Axenfeld-Rieger syndrom and other diseases involved iris abnormity were covered. The process of specific high throughput sequencing was in conformity to some published articles[12, 13].
After HiSeq 2000 sequencing, Solexa QA package and the Cutadapt program were used to filter out low quality reads and adaptor sequences and high-quality reads from raw reads were retrieved. Then the clean read sequences were aligned to the human reference genome (hg19) using SOA Paligner program. Subsequently, the single nucleotide polymorphism (SNPs) and the insertions or deletions (InDels) were identified using the SOAPsnp program and GATK program separately. The identified SNPs and InDels were annotated using ANNOVAR program (http://122. 228.158.106/exomeassistant) and viewed using MagicViewer. Finally, nonsynonymous variants were evaluated by four algorithms, Ployphen, SIFT, PANTHER and Pmut, as described previously to determine pathogenicity.
After high throughput sequencing, the detected variations were validated by Sanger sequencing in the Chinese family. Primer6.0 was used to design the PCR primer sets and the PCR products were sequenced using a Bigdye terminator v3.1 cycle sequencing kit (ABI, Foster City, CA, USA) and analyzed on an ABI 3730XL Genetic Analyzer. The primers used in this study are listed in Table 1.