2.METHOD
2.1 Ethical approval
This study was approved by the Ethics Committee of Southwest University. Written informed consent was obtained from the participants or their guardians in accordance with the guidelines of the Declaration of Helsinki.
2.2 Pedigree, proband and clinical assessment
The pedigree consisted of a proband (Fig. 1), the proband’s normal father, normal mother, normal brother and normal grandmother in a Chinese family from Luzhou, a city in Sichuan Province. For clinical assessment, a clinical history and ophthalmic examination of the proband were performed, including best-corrected visual acuity, intraocular pressure, slit lamp biological microscopy, ultrawide angle fundus imaging, optical coherence tomography, and UBM, as well as systemic examinations including heart ultrasound.
2.3 Blood sampling and DNA extraction
In this study, 2 mL of fresh peripheral blood was collected from the proband and pedigree members, and peripheral venous blood genomic DNA (gDNA) was extracted using the Wizard Genomic DNA Extraction Kit(Promega). DNA was measured using a Nanodrop 2000, and then the next experiment was carried out after quality testing criteria were met. In addition, blood samples were also taken from 100 ethnically matched and healthy control volunteers without any disease history.
2.4 Capture panel design and target sequencing
The main procedure of target region capture sequencing was using the capture chip to enrich the DNA fragments in the target region and then sequencing them with the help of a high-throughput next-generation sequencing platform. In our study, 50 known candidate genes for iris disease were captured by MyGenostics (GenCap). The process of capture was as follows: genomic DNA was randomly broken into fragments and linked to the Illumina PE junction oligonucleotide mixture. The products were amplified and purified by ligation-mediated polymerase chain reaction (LM-PCR), and then the DNA libraries were obtained and inspected. The above PCR product was hybridized with the target region capture chip to enrich the target region sequence. The captured sequences were sequenced on the Illumina HiSeq 2000 sequencing platform (Illumina, San Diego, CA, USA), and the original data were initially processed, including image recognition and sample division.
2.5 PCR amplifications and Sanger sequencing
The selected mutation sites were identified by PCR and Sanger sequencing. Primers were designed using the online software Primer 3.0. (http://primer3.ut.ee/), and the primer sequences were as follows: upstream primer, 5΄ GGGAGTGGTGCCCTACCT 3΄; downstream primer, 5΄ ACTGGTAGATGCCGTTCAGG 3΄ (Table 3). The PCR amplification reaction system was configured using the preset program of the Biomek® automatic workstation. After sufficient mixing and centrifugation, the sample was placed into a preprogrammed PCR instrument, and the program was run according to the following steps: 96°C for 1 min 30 s; 96°C for 15 s, 50°C for 6 s, and 60°C for 3 min 30 s for 28 cycles; followed by a 4°C hold. The PCR products were sequenced using Sanger sequencing and analyzed on the ABI 3130 Genetic Analyzer (Applied Biosystems), and then cosegregation analysis was performed among family members.
2.6 Protein structure and bioinformatics analysis
The original sequencing data were stripped of contamination and linker sequences and then BWA software(http://bio-bwa.sourceforge.net/) was used to compare the filtered sequences to Human Genome Reference by NCBI (hg19). GATK software (https://software.broadinstitute.org/gatk/) was used to analyze the information of single nucleotide variations (SNVs) and inserts and deletions(INDEL). All SNPs and INDELs were then annotated using ANNOVAR software (http://annovar.openbioinformatics. org/en/latest/). Mutation sites with frequencies less than 0.02 were screened out from the normal population database, which included the 1000 Genomes Project (http://www.1000genomes.org/), Exome Variant Server () and EXAC (http://exac. broadinstitute.org/).Software SIFT (http://sift.jcvi.org/), PolyPhen-2 (http://genetics.bwh.Harvard.edu/pph2/ ), MutationTaster (/) and GERP++ (http://mendel.stanford.edu/SidowLab/ downloads/gerp/index.html) software were used to predict the pathogenicity and conservatism of missense mutations, and the pathogenicity of splice site changes was analyzed using SPIDEX (http://www.deepgenomics.com/spidex) software.
2.7 Predictive analysis of the 3D model of the protein
The prediction software SWISS-MODEL (https://swissmodel.expasy.
org/interactive) was used for prediction analysis of the 3D model of the protein. The steps were as follows: input the protein sequence to be predicted and click the built model; select the model: selection covered the range of target sites; the identity and similarity of the model were not less than 30% (recommended value). The predicted model was applied using Swiss-PdbViewer (http://www.genebee.msu.su/spdbv/text/getpc.htm), a visual analysis software, and the results were obtained.