Subject recruitment and clinical examination
A four-generation family with aniridia was recruited in the Shanghai General Hospital in Shanghai, China. Thirteen family members of this family (Figure 1) took part in this study. This study was conducted in accordance with the Declaration of Helsinki and was approved by the ethics committee of Shanghai General Hospital. Informed consent was obtained from each participant. Seven of the 13 family members were diagnosed with congenital aniridia. No consanguinity was present in the family. Each family member received complete and comprehensive clinical and ophthalmic examination, including visual acuity test, intraocular pressure (IOP) measurement, anterior segment examination, slit lamp examination, fundus exam and orthoptic evaluation, as well as the examination of physical malformations and neurological deficits. In addition, 200 ethnically matched healthy individuals with no direct or collateral ties and no related phenotypes and systemic underlying diseases were recruited.
Genomic DNA was extracted from peripheral blood using the TruSeq DNA LT Sample Prep kit (Illumina, San Diego, CA) according to the manufacturer’s protocol. DNA samples were stored at −20°C until used, and DNA integrity was evaluated by 1% agarose gel electrophoresis.
Whole-genome sequencing (WGS) was performed in all 13 family members. The libraries were constructed with TruSeq Nano DNA LT Sample Prepararion Kit (Illumina, San Diego, CA, USA). Briefly, the genomic DNA was sheared into fragments with length ~350 bp using S220 Focused-ultrasonicators (Covaris, USA). Adapters were ligated onto the 3’ end of the sheared fragments. After polymerase chain reaction (PCR) amplification and purification, the final libraries were sequenced on the Illumina sequencing platform HiSeq X Ten platform (Illumina Inc., San Diego, CA, USA) and 150 bp paired-end reads were generated. The average sequencing depth was at least 30×.
Routine whole-genome sequencing analysis
The raw reads were subjected to a quality check and then filtered by fastp (https://github.com/OpenGene/fastp). Reads were aligned to hg38 using SpeedSeq . Single nucleotide variants and insertions/deletions (indels) calling were performed using Genome Analysis Toolkit v2.1 . Structural variants and copy number variants were analyzed in SpeedSeq . Annotations of single nucleotide variants, indels, structural variants and copy number variants were performed with ANNOVAR . Variant filtering was performed as illustrated in Supplementary 1.
In order to identify the exact breakpoints of the deletion in PAX6 gene after WGS analysis, primers were designed in the region surrounding the deletion using Primer3 software (version 4.0, http://bioinfo.ut.ee/primer3-0.4.0/). PCR primer pairs and amplification conditions are available upon request. PCR products were checked by 1% agarose gel electrophoresis and purified with SAP-Exon I kit (USB, USA). Purified PCR products were directly sequenced in both forward and reverse directions using an ABI 3730xl genetic analyzer (Applied Biosystems, Foster City, CA, USA) per manufacturer’s instructions. DNA sequences were analyzed using Chromas (version 2.22) and DNAMAN (version 7) software. The primer sequences were 5’-TAAATTTATTTTTGTGCTGACCTTG-3’ (forward) and 5’- ATTTCAGGCAAGTTCTGTGGTG -3 (reverse) for the PAX6 gene.