This study was performed as per the guidelines approved by the Ethics Committee of Zhongshan Ophthalmic Center (ZOC), Sun Yat-sen University and in accordance with the Declaration of Helsinki. Written informed consent was obtained from all subjects.
A 6-year-old boy complaining of gradual bilateral vision loss for the last 2 years presented in Pediatric Ophthalmology Department, ZOC. Comprehensive ophthalmic examinations were performed, including visual acuity, intraocular pressure (IOP), slit lamp microscopy, fundus photography (Heidelberg Engineering, Inc., Heidelberg, Germany), optical coherence tomography (OCT; Carl Zeiss Meditec, Inc., Dublin, CA, USA), ultrasound B scan (Aitomu Machinery Co. Ltd, Shanghai, China), and magnetic resonance imaging (MRI; General Electric, Milwaukee, WI). As choroidal ganglioneuroma was diagnosed based on previous choroidal biopsy, extensive examinations were performed to screen any systemic involvement. Whole exome sequencing was performed to unravel the genetic pathogenesis of this rare disease.
DNA sample collection
The proband was the second child of healthy parents (Figure 1). The parents had no knowledge of any cancer, or diseases associated with NF and PTEN hamartoma tumor syndrome. Blood samples were collected from the proband and his unaffected family members. A total of 200 individuals from the same population, who exhibited no diagnostic features of tumor, neurofibromatosis or PTEN hamartoma tumor syndrome, were recruited to serve as normal control population (41.99 ± 15.62 years old, 105 males/95 females). Genomic DNA was extracted using the TIANamp Blood DNA Kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s instructions. The quantity and quality of DNA were verified with NanoDrop (2000c Model, Thermo Fisher, US).
Library preparation and targeted sequencing
Illumina paired-end libraries were prepared using the Kapa LTP library prep kit (Roche, Basel, Switzerland) according to the manufacturer’s protocol. Briefly, genomic DNA was sheared into fragments approximately 300–500 bp in length. The DNA fragments were end-repaired and an extra ‘adenine’ base was added to the 3' end. Illumina adapters were ligated to the ends of the DNA fragments and subsequently four cycles of PCR amplification were performed on each sample. The DNA libraries were quantified using Qubit 3.0. Pre-capture libraries were pooled together for each capture reaction. Agilent SSELXT Human All Exon V6 was used for whole exome sequencing (Agilent, Santa Clara, CA, USA). The enriched DNA library was sequenced on Illumina Xten Analyzers, at 150 cycles per read, to generate paired-end reads following the manufacturer's standard sequencing protocols.
Bioinformatic analysis of sequencing results
Raw reads were aligned to the human genome reference (hg19) using the Burrows-Wheeler Aligner. Single-nucleotide variants (SNVs) and insertions and deletions (InDels) were called by GATK4.0 HC. The frequency of all SNVs and InDels was annotated using the ExAC, gnomAD, HGVD, CHARGE, 1000 Genome, UK10K databases, and the internal database of Clinbytes Inc. to filter the common variants, with an allele frequency cutoff of 0.5% and 0.1% for recessive and dominant variants, respectively.
After the confirmation of pathological variants in the proband, samples from all available and consenting family members were Sanger sequenced for segregation analysis. PCR primer sets were designed via Primer3 and products were sequenced on an ABI 3700XL Genetic Analyzer. The sequences of forward and reverse primers used for amplification of PTEN are 5’-GGCTACGACCCAGTTACCATAG-3’ and 5’-TGGGACAGGTTCTTCCATCATC-3’, respectively.