Subjects and Clinical Evaluations
To make a precise diagnosis, we performed targeted next-generation sequencing (NGS) of microphthalmos-related genes. Written informed consent of the family members from both families was obtained prior to the collection of 5 mL of their peripheral blood for the following experiment. The study was approved by the ethics committee of Eye and ENT Hospital of Fudan University and was conducted according to the principles of the Declaration of Helsinki.
Our study involved two family members from three generations who underwent detailed history ophthalmic examination, including best corrected visual acuity (BCVA) testing, slit lamp biomicroscopy, IOL master 500 (Carl Zeiss Meditec, Germany), dilated fundus examination, B scan, and SD-OCT (Spectralis HRA + OCT, Heidelberg, Engineering, Inc., Heidelberg, Germany). Family and medical history, including age of onset and other related clinical manifestations, were obtained. Blood samples were collected from the peripheral blood and stored at 4 °C before further analysis.
Genomic DNA from the family was extracted from peripheral blood on Feb 2018 according to the manufacturer's standard procedure using the QIAamp DNA Blood Midi Kit (Qiagen, Hilden, Germany) (Ma et al. 2015). A capture panel (NimbleGen, Madison, USA) of microphthalmos genes has been previously designed and assessed by our group. The capture panel comprised all exons together with the flanking exon and intron boundaries (±15 bp) of 425 genes that are most frequently involved in common inherited eye diseases. The capture probes (callinonel, scall-in-one) were designed by BGI and produced by Roche.
The library was enriched by array hybridization according to a previously published procedure, followed by elution and post-capture amplification. Then, qualification and NGS targeted sequences were further analyzed on the Illumina HiSeq 2000 platform (Illumina, Inc., San Diego, CA, United States) in collaboration with BGI-Shenzhen (Shenzhen, Guangdong, China) as previously reported (Chen et al. 2015, 2013; Qi et al. 2017). To detect the potential variants in the family, we performed bioinformatics processing and data analysis after receiving the primary sequencing data. We used previously published filtering criteria to generate “clean reads” for further analysis. The “clean reads” (with a length of 90 bp) derived from targeted sequencing and filtering were then aligned to the human genome reference (hg19) using the Burrows–Wheeler Aligner (BWA) Multi-Vision software package (Li and Durbin 2009). After alignment, the output files were used to perform sequencing coverage and depth analysis of the target region, single-nucleotide variants (SNVs) and INDEL calling.
We used the following four databases to test annotation of all identified variants with minor allele frequency (MAF) > 0.1% to eliminate benign variants: dbSNP137, HapMap Project, 1000 Genomes Project and Exome Variant Server. Finally, the variant prioritizations were performed, combining total depth, quality score, MAF, potential deleterious effect and the existence of mutation reports in common databases, such as the Human Gene Mutation Database (HGMD) and Online Mendelian Inheritance in Man (OMIM). All mutations and potential pathogenic variants were validated using the conventional Sanger sequencing or spectrum sequencing method. Segregation analysis was performed if DNA from family members was available.
For Sanger verification sequencing, primers were designed on the upstream and downstream of the fragment for all the potential pathogenic mutations. PCR amplification was carried out and Sanger sequencing was done, then we compared the result with the standard sequence of PXDN and CRYBB2 genes to verify the result of next-generation sequencing-based target capture sequencing.
We used the spectrum sequencing method to validate the variant if the common allele was detected by the Sanger sequencing. Agena MassARRAY platform was used for genotyping. It is a robust tool with high accuracy and is cost effective as it involves multiplex PCR and single-base extension. The high-energy laser could make the extended oligonucleotide product have a single charge. The genotype of the corresponding locus could be judged based on different flight time of different oligonucleotides under the action of an electromagnetic field.