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.