New mutation in PTEN identied in patient with rare bilateral choroidal ganglioneuroma

Background: Choroidal ganglioneuroma is an extremely rare tumor, with little known regarding its pathogenesis. The present study aimed to investigate the phenotype and genetic alterations in one sporadic patient with rare bilateral choroidal ganglioneuroma. Methods: A 6-year-old boy with histological diagnosis of bilateral ganglioneuroma was recruited in this study. Comprehensive ophthalmic examinations were performed in the patient. Genomic DNA was extracted from peripheral blood collected from the patient, the patient's unaffected family members, and from 200 unrelated control subjects from the same population. Whole exome sequencing was carried out and raw reads were aligned to the human genome reference (hg19) using the Burrows Wheeler Aligner. All available family members were subjected to Sanger sequencing for segregation analysis. Results: Bilateral and extensive retinal detachments were observed in OCT. The diffuse thickening of choroid was identied in B scan and MRI. Genetic analysis revealed the presence of a novel heterozygous PTEN frameshift mutation, c.498delA (p.Thr167LeufsTer16), in exon 6. It was identied in the affected individual, but not in any of the unaffected family members. Genetic analysis showed that there was no mutant in neurobromatosis-related genes. There were no obvious abnormalities in other organs in comprehensive systemic examinations. Conclusions: A novel de novo PTEN mutation was identied in a bilateral choroidal ganglioneuroma case. Although PTEN mutation has been considered to induces multiple abnormalities, choroidal ganglioneuroma can be the rst manifestation without abnormalities in other organs. Further studies are needed to conrm this new association between choroidal ganglioneuroma and the PTEN mutation.

ganglioneuroma can be the rst manifestation without abnormalities in other organs. Further studies are needed to con rm this new association between choroidal ganglioneuroma and the PTEN mutation.
According to the previous reports, blind painful eye was the common feature, and 12 cases have established neuro bromatosis type 1 (NF-1), which lead to the suggestion that choroidal ganglioneuromas could be recognized as a rare manifestation within the clinicopathologic spectrum of NF-1 syndrome 19 . Recently, we confronted with the rst case of bilateral choroidal ganglioneuromas, and reported the multimodal imaging features in the early stage 20 .
Aiming to unravel the pathogenesis of this rare disease, whole exome sequencing was carried out in this patient. As a phosphatase and tensin homolog (PTEN) mutation was identi ed, comprehensive systemic examinations were performed to screen the presence of concomitant abnormalities.

Study participants
This study was carried out according to 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.

Library preparation and targeted sequencing
According to the manufacturer's protocol, Illumina paired-end libraries were prepared using Kapa LTP library prep kit (Roche, Basel, Switzerland). Brie y, genomic DNA was sheared into fragments of 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 four cycles of PCR ampli cation were applied to each sample after ligation. The DNA libraries were quanti ed by the Qubit 3.0. Pre-capture libraries were pooled together for one 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 for 150 cycles per read to generate paired-end reads following the manufacturer's standard sequencing protocols.

Bioinformatics 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 were annotated using the ExAC, gnomAD, HGVD, CHARGE, 1000 Genome, UK10K databases, and the internal database of Clinbytes Inc. to lter the common variants, with an allele frequency cutoff of 0.5% and 0.1% for recessive and dominant variants, respectively.

Genetic validation
After any pathologic variants were con rmed in the proband, samples from all available family members were subjected to Sanger sequencing for segregation analysis. PCR primer sets were designed via Primer3 and products were sequenced on an ABI 3700XL Genetic Analyzer. Primers used for ampli cation of PTEN are forward 5'-GGCTACGACCCAGTTACCATAG-3' and reverse 5'-TGGGACAGGTTCTTCCATCATC-3'.

Ocular ndings
In initial examination in 2 years ago, the best corrected visual acuity was 20/50 in the right eye and 20/32 in the left. In recent visit, it was hand motion/10 cm and 20/200, respectively. The IOP had uctuated around the normal range in the past, but 40 mmHg IOP was observed in recent visit, and was controlled well with brinzolamide-timolol eyedrops.
Bilateral and extensive retinal detachments were observed in OCT. The diffuse thickening of choroid was identi ed via B scan and MRI ( Figure 2). Choroidal tumor was highly suspected, and following choroid biopsy demonstrated the histological diagnosis of choroidal ganglioneuroma. More details on the multimodal images can be found in our previous report 20 .

Mutation screening
Genetic analysis documented the presence of a heterozygous PTEN frameshift mutation, c.498delA (p.Thr167LeufsTer16) in exon 6 (NM_000314). It was identi ed in the affected individual, but not in any of the unaffected family members; therefore, this was considered a de novo mutation. The identi ed mutation had not previously been reported, and was not observed in a large population cohort (Figure 3). The c.498delA variant causes a frameshift starting with codon Threonine167, changing this amino acid to a Leucine residue, and creates a premature Stop codon at position 16 of the new reading frame, denoted p.Thr167LeufsTer16. This variant is predicted to cause loss of normal protein function either through protein truncation or nonsense-mediated mRNA decay.
As most reported cases showed a medical history of NF-1, genetic changes in neuro bromatosis-related genes were evaluated. Genetic analysis showed that there was no mutant in NF1-or NF2-related genes.

Systemic workup
According to histological nding of ganglioneuromas and the mutation in PTEN, it is crucial to rule out the presence of ganglioneuromas, or PTEN related abnormalities in other tissues. Comprehensive systemic examinations were performed in the case, including general physical examination, ultrasound scan of the thyroid gland, liver and kidney, CT scan of the mediastinum and retroperitoneum, and no obvious abnormalities were detected.

Discussion
According to the reported studies (Table 1), most cases of choroidal ganglioneuroma share the same features. First, most cases of uveal ganglioneuroma co-occurred with NF-1, leading to the diagnosis as NF-1with orbit-facial involvement 19 . Second, ganglioneuroma leads blind and painful eye unilaterally, and end up with evisceration/enucleation in all cases. Third, ganglioneuroma were diagnosed unexpected only after subsequent histopathological examination. Genetic examination was performed in only one case diagnosed as Cowden syndrome and revealed a PTEN mutation. Although the pathogenic gene has been identi ed, the mechanisms are still unknown yet that why it causes bilateral choroidal ganglioneuroma, without any obvious changes throughout the whole body.
This may be attributed to the young age of this patient. Studies have reported features with respect to age of onset in PTEN mutation carriers. For instances, the onset age of breast cancer of PHTS is believed to be around age 38-50 years, and average age of diagnosis are in the 40s for PHTS related cancers in thyroid, endometrial and colon [28][29][30] . Thus, genetic study is important the patients, as PTEN mutation has been considered to increase risks for multiple common cancers. Lifetime cancer risk estimates and close follow-up are highly required for the young boy in the present case.
One limitation of the present study is the limited case number. Choroidal ganglioneuroma is an extremely rare disease and most reported cases are not diagnosed until evisceration/enucleation. Therefore, it might be very di cult to recruit enough cases for a case-series study. Written informed consent for the publication of these details have been obtained from the patient's patient.

Availability of data and materials
All the data used to support the ndings of this study are included within the article and are available from corresponding author by a reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
This study was supported in part by grants from the Fundamental Research Funds of State Key Laboratory of Ophthalmology, research funds of Sun Yat-sen University (15ykjc22d; Guangzhou, Guangdong, China), and Science and Technology Program Guangzhou, China (201803010031; Guangzhou, Guangdong, China). The sponsors and funding organizations had no role in the design or conduct of this research.

Authors' Contributions
DXY conceived and designed the study. JZX, ZT, CCL and LSS participated in data collection, laboratory analysis and interpretation. JZX and SLM analyzed the data and wrote the rst draft of the manuscript. DXY and CCC critically reviewed the manuscript. All authors approved the submitted version.