Genetics Detection of Clinically Relevant Genetic Variants in Chinese Patients With Nanophthalmos by Trio-Based Whole-Genome Sequencing Study

Congcong Guo,1 Zhenni Zhao,1 Denghui Chen,1 Shuxiang He,2 Nannan Sun,1 Zhongwen Li,1 Jiafan Liu,1 Dandan Zhang,1 Jiamin Zhang,1 Jianlong Li,1 Miao Zhang,1 Jian Ge,1 Xing Liu,1 Xiaoling Zhang,3,4 and Zhigang Fan1 1State Key Laboratory of Ophthalmology, Department of Glaucoma, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China 2Guangzhou KingMed Diagnostics, Guangzhou, China 3Section of Biomedical Genetics, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, United States 4Department of Biostatistics, Boston University School of Public Health, Boston, Massachusetts, United States

Correspondence: Zhigang Fan, State PURPOSE. Nanophthalmos is a rare genetic disorder commonly characterized by a short axial Key Laboratory of Ophthalmology, length (AL) and severe hyperopia. Mutations that have been identified through Mendelian Department of Glaucoma, Zhong- genetic analysis can only explain a fraction of nanophthalmic cases. We investigate the shan Ophthalmic Center, Sun Yat-sen clinically relevant genetic variants in nanophthalmos by whole-genome sequencing (WGS), University, Guangzhou 510060, including de novo mutations (DNMs) and inherited mutations. China; [email protected]. METHODS. Clinically relevant genetic variants of 11 trios (11 nanophthalmic probands and Xiaoling Zhang, Section of Biomedi- their unaffected parents) from the Zhongshan Ophthalmic Center, China, were analyzed by cal Genetics, Department of Medi- WGS. We further screened three trios and 10 sporadic cases to identify the MYRF mutations. cine, Boston University School of Medicine, 72 East Concord Street, RESULTS. In two of 11 trios, without evidence of the presence of deleterious inherited Room E223, Boston 02118-2526, MA, autosomal variants, two DNMs of MYRF (c.789delC, p.S264fs and c.789dupC, p.S264fs) were USA; identified in the probands. These loss-of-function DNMs were predicted to result in premature [email protected]. stop codons and protein structure damage in both probands. In addition, deleterious CG and ZZ contributed equally to the inherited genetic variants in PRSS56 and MFRP were found in eight probands of the other work presented here and should nine trios. Expanded screening found an additional MYRF DNM (c.1433G>C, p.R478P) in therefore be regarded as equivalent one trio and a stop-gain MYRF mutation (c.2956C>T, p.R986X) in one sporadic case, authors. suggesting the recurrence of MYRF mutations in nanophthalmic patients. Submitted: November 25, 2018 CONCLUSIONS. This is the first trio-based WGS study for nanophthalmos, revealing the potential Accepted: May 23, 2019 role of DNMs in MYRF and rare inherited genetic variants in PRSS56 and MFRP. The Citation: Guo C, Zhao Z, Chen D, et al. underlying mechanism of MYRF in the development of nanophthalmos needs to be further Detection of clinically relevant genetic investigated. variants in Chinese patients with Keywords: de novo mutation, MYRF, nanophthalmos, trio analysis, whole-genome sequencing nanophthalmos by trio-based whole- genome sequencing study. Invest Ophthalmol Vis Sci. 2019;60:2904– 2913. https://doi.org/10.1167/ iovs.18-26275

anophthalmos and posterior microphthalmos are two to have a strong genetic basis. With the availability of next- N subtypes of microphthalmos, which are bilateral small generation sequencing technology, genetic testing for nano- eyes derived from developmental defects.1 The estimated birth phthalmos has been improved substantially.8 Recent sequenc- prevalence for microphthalmos is 0.002% to 0.017% worldwide ing studies of whole exomes and candidate genes have revealed and 0.009% in China.2,3 Nanophthalmos is a relatively rare some of the molecular mechanisms responsible for certain condition characterized by a short axial length (AL; commonly patients with nanophthalmos.6,8,9 Nanophthalmos 1 (NNO1, <20 mm) due to small anterior and posterior segments, a thick Online Mendelian Inheritance in Man [OMIM] 600165) at 11p choroid and sclera, and marked hyperopia.4–6 Due to their was the first human locus identified to be associated with specific anatomic features, nanophthalmic patients are prone to nanophthalmos.10 Subsequently, studies in a series of nano- develop a crowded anterior chamber and eventually angle- phthalmos pedigrees have detected transmembrane protein 98 closure glaucoma.7 If not diagnosed and treated, angle-closure (TMEM98, OMIM 615949) at 17q11,6,9 membrane-type frizzled- glaucoma can progress to optic nerve damage and blindness. related protein (MFRP, OMIM 606227) at 11q23,11 serine Nanophthalmos is a consequence of the developmental protease 56 (PRSS56, OMIM 613858) at 2q37,12,13 crumbs arrest of eyes in the early stages of embryogenesis. It is thought homolog 1 (CRB1, OMIM 604210) at 1q31,14,15 bestrophin 1

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(BEST1, OMIM 607854) at 11q12,16 and nanophthalmos 3 prepare the WGS library, followed by enzymatic fragmentation (NNO3, OMIM 611897) at 2q11-q14.17 Most recently, Garnai et with end-repair/A-tailing and ligation. Adapter-ligated DNA was al.18 established myelin regulatory factor (MYRF,OMIM purified using AMPure XP beads, and the resultant fragments 608329) as a novel nanophthalmos gene in the Caucasian were amplified for eight cycles of PCR. The final DNA library population. Nanophthalmos may present as a sporadic or was purified with AMPure XP beads, and then the library familial condition, with autosomal-recessive or -dominant concentration was quantified using a Qubit fluorometer inheritance.19 According to the OMIM database, autosomal- (Thermo Fisher Scientific, Waltham, MA, USA). The libraries dominant genes include NNO1, NNO3, TMEM98 (NNO4), were then sequenced using the Novaseq 6000 sequencer BEST1, and MYRF, and autosomal-recessive genes include (Illumina, San Diego, CA, USA) at KingMed Diagnostics PRSS56, CRB1, and MFRP (NNO2). However, taken together, (Guangzhou, China). The sequencing depth in subjects of the these genetic mutations identified through classic Mendelian discovery stage ranged from 44.813 to 71.063 (proportion of genetic analysis can only explain a fraction of nanophthalmos sequencing depth >103 per sample ranged from 91.48% to cases. 92.28%). The Q20 (1 error in 100) read quality score ranged In terms of whole-exome sequencing (WES), it depends on from 93.77% to 95.87%, and the Q30 (1 error in 1000) ranged different exon-capture kits/platforms, which might miss some from 86.00% to 92.11%. The mapping rate of clear data ranged exon variants. In addition, the exon-capture kits were designed from 99.44% to 99.88%, and the genome coverage ranged from based on the available human gene/exon annotation database, 91.73% to 92.38% (Supplementary Table S1). which has been updating all the times. Whole-genome The sequencing data were processed with Burrows- 22 sequencing (WGS) will not have these issues. By comparison Wheeler Aligner (BWA) for alignment on a reference with WES, WGS encompasses a greater number of annotated sequence of hg19 and the GATK-HaplotypeCaller package 20 (GATK version 3.5-0-g36282e4; Broad Institute, Cambridge, exons with coverage sufficient for variant calling. In this 23–25 study, to investigate the novel genetic mechanism potentially MA, USA) for local realignment, quality recalibration, and variant calling. Then, the Variant Call Format (VCF) files were explaining other cases of nanophthalmos, we conducted a trio- 26 based WGS study for nanophthalmos, representing, to the best analyzed using the ANNOVAR tools. The number of single of our knowledge, the first report with this specific purpose. nucleotide variants (SNVs) and small insertions/deletions (indels) per subject is summarized in Supplementary Table S1. Potential Mendelian violations were identified if there was a METHODS heterozygous genotype in the proband and homozygous wild- type genotype in the unaffected parents.20 After eliminating Description of the Participants the low-quality variants by GATK’s filters, Mendelian violations ranged from 51,151 to 151,542 in 11 trios (Supplementary This study was approved by the Human Research Ethics Table S1). Then these variants were filtered using the following Committee of Zhongshan Ophthalmic Center, Sun Yat-Sen criteria: (1) sequence coverage more than 103; (2) frequency University (Guangzhou, China) and adhered to the tenets of in 1000 Genome Project (1000G), dbSNP137 and Exome the Declaration of Helsinki. All subjects provided written Aggregation Consortium (ExAC) less than 1%; (3) nonsense informed consent. In the discovery stage, we recruited 11 trios mutation, frameshift mutation, splice site, or predicted with a nanophthalmos proband and unaffected parents. The damaging missense mutation; and (4) 35% to 65% of reads unrelated probands consisted of seven males and four females, were the reference call. Finally, a total of 15 de novo mutations and the mean age at recruitment was 30.09 6 12.14 (11–48)- (DNMs) were detected in 11 probands (1.36 DNMs per years old. In the validation stage, three additional trio families proband), which were consistent with published papers27,28; and 10 sporadic cases with mean age of 42.15 6 13.43 (15– of these variants, eight were splice sites, four were missense 63)-years old were recruited for expanded screening of MYRF mutations, two were frameshift mutations, and one was a gene. The validation cohort consisted of six males and seven synonymous variant (Supplementary Table S2 and S3). The females. The diagnosis of nanophthalmos was made by a short analysis workflow is shown in Figure 1. AL (<20 mm) and the absence of morphologic malformation. We determined the inherited autosomal variants by All eligible individuals underwent the following ophthalmic comparing the genotypes of probands and parents. Rare evaluations: best-corrected visual acuity (BCVA), slit-lamp variants (frequency <0.1%) were filtered to maintain only examination, IOP measurement (Goldmann applanation to- deleterious variants, including nonsense mutations, frameshift nometry), and fundus examination. The AL was measured by A- mutations, splice sites, or predicted damaging missense scan ultrasonography (Quantel Medical, Cournon d’Auvergne mutations (based on predictive programs, such as SIFT, Cedex, France). Anterior chamber depth (ACD) and anterior PolyPhen-2,MutationTaser,VEST3,MCAP,CADD,Eigen, chamber angle were examined with an ultrasound biomicro- FATHMM, and GenoCanyon; Supplementary Table S4). Addi- scope (UBM; Paradigm Medical Industries, Salt Lake City, UT, tionally, the American College of Medical Genetics and USA). The angle grading of all probands were IV by gonioscopy. Genomics (ACMG) guidelines29 (Table 1) and minor allele Subjects with uncontrolled ocular infection, severe systemic frequency of each variant in the Genome Aggregation Database diseases, or lens nucleus of grade 4 or harder according to the (GnomAD)30 (Table 2) were employed. Emery-Little classification,21 or who declined to participate were excluded. Expanded screening to Identify MYRF Mutations in Trios and Sporadic Cases Trio-Based Whole-Genome Sequencing Additional three trio families and 10 sporadic nanophthalmic Peripheral blood samples were obtained from all subjects, and patients were recruited for expanded screening of MYRF gene. genomic DNA was extracted using an isolation kit (OSR-M104- WGS was also performed in the additional trios. The T1; Tiangen Biotech, Beijing, China) according to the sequencing depth in these nine samples ranged from 47.443 manufacturer’s instructions. The DNA was sheared into to 66.213 (proportion of sequencing depth ‡ 103 ranged from fragments of 350 bp using a Covaris LE220 Focused Ultra- 91.55%–92.27%). The Q20 ranged from 93.89% to 94.92%, and sonicator (Covaris, Woburn, MA, USA). The NEXTflex Rapid the Q30 ranged from 86.29% to 92.30%. The mapping rate of DNA-Seq Kit (BIOO Scientific, Austin, TX, USA) was used to clear data ranged from 99.55% to 99.72%, and the genome

Downloaded from iovs.arvojournals.org on 10/04/2019 A Trio-Based WGS Study for Nanophthalmos IOVS j July 2019 j Vol. 60 j No. 8 j 2906 AL, mm ACD, mm BCVA IOP, mm Hg OD OS OD OS OD OS OD OS ACMG Evidence Sex Age, y

FIGURE 1. Flow chart of the discovery of the de novo mutations in nanophthalmos by WGS. The numbers in parentheses represent the range of the number of variants at each stage in 11 trios in the discovery stage. aA Mendelian violation was identified if there was a heterozygous genotype in the proband and homozygous wild-type genotype in both parents; and low-quality variants were eliminated ACMG b

Category with GATK’s recommended filters. Deleterious variants included nonsense mutations, frameshift mutations, splice sites, or predicted damaging missense mutations. cZero DNMs from one trio, one DNM from seven trios, two DNMs from two trios, and four DNMs from one trio.

Variants coverage ranged from 91.76% to 92.38% (Supplementary Table S1). WES at an average sequencing depth of 106.393 (range 92.413 to 122.193) was performed in the further 10 sporadic MYRF cases by using Novaseq 6000 sequencer (Illumina). The detailed statistical information of the WES data was shown in Supplementary Table S5. C, p.R478P Missense Likely pathogenic PS2, PM2, PP3 F 35 17.76 18.02 0.5CT 2.37 NLP 0.50 38.00 14.00 T, p.R986X Stop-gain Likely pathogenic PVS1, PM2 M 32 17.00 17.00 3CT – 0.63 0.50 12.00 13.00 > > Sanger Sequencing of Target Regions for Validation Amplification using PCR followed by Sanger sequencing was used to confirm the potential DNMs of MYRF in each family member of Trio numbers 8, 10, and V1 (NM_001127392: c.789delC, c.789dupC and c.1433G>C) and the stop-gain MYRF mutation in sporadic patient number 2 (NM_001127392: c.2956C>T). Sequencing was performed on an ABI 3730xl DNA analyzer (Applied Biosystems, Foster Clinical Characteristics of Patients With City, CA, USA). The reference genes were obtained from the

1. Ensembl genome browser (in the public domain, http://www. CT, corneal thickness; del, deletion; dup, duplication; fs, frameshift; F, female; M, male; NLP, no light perception. Patient Number Change Mutation Type ensembl.org). The primers used for PCR amplification are ABLE 8 (Trio number 8)10 (Trio number 10)V1 (Trio number V1) c.789delC, c.789dupC, p.S264fs p.S264fs c.1433G Frameshift Frameshift duplication deletion Pathogenic Pathogenic PVS1, PS2 PVS1, PS2 F F 30 16 17.58 17.33 18.09 18.07 2.76 2.68 2.49 0.10 2.37 0.50 0.02 0.20 13.70 27.00 13.20 24.00 T Sporadic case number 2 c.2956C listed in Supplementary Table S6.

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TABLE 2. The Minor Allele Frequencies in the GnomAD Database for the Variants Found in Nanophthalmic Patients

Patient Number Gene Mutation MAF in GnomAD

1 (Trio number 1) PRSS56 c.1066dupC, p.Gln356fs (het) 58/150520 c.1687delC, p.Arg563fs (het) 0 2 (Trio number 2) PRSS56 c.343dupG, p.Ala115fs (het) 1/97248 c.1508T>A, p.Met503Lys (het) 0 4 (Trio number 4) PRSS56 c.1066dupC, p.Gln356fs (het) 58/150520 c.781G>A, p.Gly261Arg (het) 2/127322 5 (Trio number 5) MFRP c.577_578delAG, p.Ser193fs (hom) 0 6 (Trio number 6) PRSS56 c.1186G>A, p.Glu396Lys (het) 0 c.1183þ32_1186þ50del (het) 0 7 (Trio number 7) PRSS56 c.1066dupC, p.Gln356fs (het) 58/150520 c.1186G>A, p.Glu396Lys (het) 0 8 (Trio number 8) MYRF c.789delC, p.S264fs (het) 0 9 (Trio number 9) MFRP c.661C>T, p.Pro221Ser (het) 3/226800 c.1411G>A, p.Val471Met (het) 4/243284 10 (Trio number 10) MYRF c.789dupC, p.S264fs (het) 0 11 (Trio number 11) MFRP c.498delC, p.Asn167fs (hom) 31/274316 V1 (Trio number V1) MYRF c.1433G>C, p.R478P (het) 0 Sporadic case number 2 MYRF c.2956C>T, p.R986X (het) 0 MAF, minor allele frequency; het, heterozygous; hom, homozygous.

Paternity and Maternity Tests RESULTS Paternity and maternity tests were used to determine the The analysis workflow of the discovery of the DNMs in the biological relationships between parents and probands in Trio WGS is shown in Figure 1. Among the total of 11 trios in the 8, 10, and V1. Genomic DNA isolation was carried out for each discovery stage, by comparing probands with their unaffected family member according to ‘‘Specifications for examination in parents, two DNMs in MYRF at 11q12.2 were identified in two forensic DNA laboratory’’ (GA/T 383-2014). 21Plex Short probands. Patient number 8 (trio number 8) was a 16-year-old Tandem Repeat (STR) Fluorescence DNA Detection Kit girl from Henan Province, China with bilaterally short AL (OD: (Jiangsu Superbio Biomedical Co., Ltd., Nanjing, China) were 18.09 mm; OS: 18.07 mm). She had poor eyesight from an early used to determine genetic characteristics. Twenty-one STR loci age and was diagnosed with amblyopia at the age of 7 years. (D3S1358, D1S1656, D6S1043, D13S317, Penta E, D16S539, When she was 15-years old, her vision gradually diminished. D18S51, D2S1338, CSF1PO, Penta D, TH01, vWA, D21S11, Ophthalmic examinations showed that her BCVAs were 0.02 D7S820, D5S818, TPOX, D8S1179, D12S391, D19S433, FGA, (þ8.75 diopters [D]) OD and 0.2 (þ7.00 D) OS. Her IOP was 27- and Amelogenin) were analyzed. The products were separated mm Hg OD and 24-mm Hg OS. UBM showed that her ACD was and analyzed on ABI Prism 3100 Genetic Analyzer (Applied 2.68-mm OD and 2.37-mm OS. Patient number 10 (trio number Biosystems). Data were analyzed using GeneMapper ID 10) was a 30-year-old woman from Guizhou Province, China software (Applied Biosystems). In parallel, appropriate positive with bilaterally short AL (OD: 17.58 mm; OS: 17.33 mm). She and negative controls were performed and confirmed to give had blurred eyes for more than 20 years and had experienced a the expected and correct results for each sample submitted. rapid decline in vision in her right eye when she was 30-years old. Her BCVAs were 0.1 (þ11.00 D) OD and 0.5 (þ11.75 D) Bioinformatics Analyses OS. UBM showed that her ACD was 2.76 mm OD and 2.49 mm OS. Both patients were diagnosed with secondary angle- We performed a gene ontology (GO) analysis using the closure glaucoma when they came to our department. The Database for Annotation, Visualization, and Integrated Discov- clinical characteristics of the two patients are shown in Table ery (David, in the public domain, https://david.ncifcrf.gov/) 1. (Supplementary Table S7). The SWISS-MODEL (in the public Two novel DNMs in MYRF (NM_001127392) were con- domain, https://swissmodel.expasy.org/) was used to predict firmed by Sanger sequencing (c.789delC (p.S264fs) in trio the impact of DNMs on the protein structure of MYRF. number 8 and c.789dupC (p.S264fs) in trio number 10, see Evolutionary conservation analysis of altered amino acid Figs. 2A–2B). The sequencing depth for the two mutations in residues was performed by comparisons across different the probands is 933 and 643, with 51.6% and 54.7% reads species (in the public domain, https://www.ncbi.nlm.nih. were mutation calls, respectively. By referring to the standards gov/protein/MYRF). and guidelines from the ACMG, those two DNMs were classified as ‘‘pathogenic,’’ based on PVS1 (frameshift) and Gene Coexpression and Genetic Interaction PS2 (de novo).29 Biological relationships between parents and Network Analyses probands were confirmed (Supplementary Tables S9 and S10). Both of these DNMs are loss-of-function variants (frameshift). To build the gene coexpression and genetic interaction None of the DNMs were found in the following publicly networks, we reviewed the PubMed database to look for population databases, including GnomAD (Table 2), Exome candidate genes related to nanophthalmos,31 microphthalmia, Sequencing Project 6500 (ESP6500siv2), and 1000G. Although anophthalmia, and coloboma (MAC),32–34 high hyperopia,35 the DNMs in MYRF have been a given dbSNP reference SNP ID angle-closure glaucoma,36 and AL37 (Supplementary Table S8). (rs774654800 and rs769274302; and rs774654800 was merged Coexpression and genetic interaction networks were created into rs769274302, Build 151), they occur at very low and visualized by GeneMANIA in the Cytoscape program.38,39 frequencies (4.05 3 105 and 1.62 3 104, respectively) in

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FIGURE 2. Identification of mutations in the MYRF gene by Sanger sequencing. (A) The DNM (MYRF: c.789delC, p.S264fs) in trio number 8. (B) The DNM (MYRF: c.789dupC, p.S264fs) in trio number 10. (C) The DNM (MYRF: c.1433G>C, p.R478P) in trio number V1. (D) The stop-gain MYRF mutation (c.2956C>T, p.R986X) in sporadic case number 2. (E) Schematics of the functional domains of the MYRF protein and locations of the MYRF mutations. P, proband; F, father; M, mother. The square represents male and circle represents female; black represents nanophthalmic patients and white represents unaffected parents.

the Asian population according to the ExAC database. Thus, Figure 6. Among them, p.Gln356fs in PRSS56 and p.Asn167fs the theoretical probability of two DNMs in 11 individuals is as in MFRP have been reported in nanophthalmos cases.42,43 low as 7.20 3 107. Through sequence alignment and protein In the validation stage, we further screened the MYRF structure prediction, we found that the de novo loss-of- mutations in three trios and 10 sporadic cases. Interestingly, function (frameshift) mutations in MYRF resulted in premature we also identified one MYRF DNM (c.1433G>C, p.R478P) in stop codons (patient number 8: codon 271; patient number 10: trio number V1, which was confirmed by Sanger sequencing codon 337) and protein structure damage in both patients (Fig. (Fig. 2C). This DNM was classified as ‘‘likely pathogenic,’’ 2E). Evolutionary conservation analysis showed that the based on PS2, PM2, and PP3 following the ACMG guidelines.29 impaired amino acid residues were mostly evolutionarily Biological relationships between parents and proband were conserved, indicating that the mutation was likely pathogenet- confirmed (Supplementary Table S11). Multiple lines of ic (Fig. 3A). computational evidence support a deleterious effect (Supple- To further examine the relationships of MYRF with genes mentary Table S4). Additionally, we also identified a MYRF associated with nanophthalmos, MAC, AL, high hyperopia and stop-gain mutation (c.2956C>T, p.R986X) in a sporadic nano- angle-closure glaucoma (Supplementary Table S8), gene coex- phthalmic patient, which was then confirmed by Sanger pression, and genetic interaction network analyses were sequencing (Fig. 2D). Based on PVS1 and PM2 following the performed (Fig. 4). Among these networks, MYRF was ACMG guidelines, this mutation was classified as ‘‘likely coexpressed with TMEM98 (nanophthalmos-related gene), pathogenic.’’ Both of these MYRF mutations found in the trio RSPO1 (AL-related gene), BMP4 (MAC-related gene), and and the sporadic case were absent from the GnomAD, 40,41 PITX3 (MAC-related gene). Genetic interaction network ESP6500siv2, 1000G, and ExAC databases. Evolutionary con- analysis indicated that MYRF interacted with GLIS3 (angle- servation analysis showed that the mutations were mostly closure glaucoma-related gene). evolutionarily conserved (Figs. 3B, 3C). The clinical character- The inherited autosomal variants in the 11 trios are shown istics of the two patients are shown in Table 1. Taken together, in Figure 5 and Table 3, and the description of probands is the recurrence of the MYRF mutations in both trio and summarized in Supplementary Text S1. Given that all the sporadic case supported the potential pathogenicity role for parents were unaffected, the autosomal mutations were limited MYRF gene in Chinese nanophthalmos patients. to the model of recessive inheritance, including PRSS56 and MFRP. We found that eight of 11 (72.7%) families carried rare mutations that segregated in an autosomal-recessive manner DISCUSSION with deleterious effects. These low-frequency PRSS56 and MFRP mutations were predicted to be damaging in five and Our study presents the first trio-based WGS analysis of three families, respectively (Fig. 5; Tables 2, 3). The detailed nanophthalmos, a rare genetic ocular disorder. Nanophthalmos locations of all variants in PRSS56 and MFRP are shown in (dwarf eye) can be regarded as an extreme developmental

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spectrum of AL and chamber angle. The study of nano- phthalmos in Chinese patients would have important implica- tions for understanding the development of AL, shallow anterior chamber, narrow anterior chamber angle, and angle- closure glaucoma in Asian populations. Furthermore, the regulatory mechanism of AL would also likely be beneficial for the study of myopia, as the mechanism responsible for ‘‘short eye’’ likely colocalizes with pathways that are involved in longer eyes (myopia). In addition to these clinical features in the anterior segment, the choroid and sclera are thicker in nanophthalmic than healthy eyes.44,45 Thus, the underlying mechanism of nanophthalmos can be exploited for the study of both anterior and posterior segments. In the present study, we discovered two novel frameshift DNMs (c.789delC, p.S264fs and c.789dupC, p.S264fs) in the MYRF gene in two Chinese nanophthalmos probands, and identified one different MYRF DNM (c.1433G>C, p.R478P) and one MYRF stop-gain muta- tion (c.2956C>T, p.R986X) in the expanded screening of trios and sporadic nanophthalmic patients. Our trio-based WGS and two-stage screening support that MYRF might be a potentially nanophthalmos gene, which is consistent with the latest research using a linkage analysis and pooled sequencing approach by Garnai et al.18 MYRF is a constrained gene that is intolerant to loss of function mutations according to the ExAC database (pLI ¼ 1), which may be caused by depletion of haploinsufficiency.30 Northern blot analysis revealed a high level of MYRF in a retinal pigment epithelial cell line.46 BioGPS (in the public domain, http://bi ogps.org) and SAGE (in the public domain, https://cgap.nci. nih.gov/) also indicated a high mRNA expression of MYRF gene in normal retinal tissues. The retina is important for normal emmetropization, which optimizes the image focus in response to visual experience by adjusting the axial dimensions.47 Whether MYRF participates in this postnatal process is unknown. It has previously been reported that MYRF is responsible for central nervous system (CNS) myelination and may regulate oligodendrocyte (OL) differentiation, which contains a DNA-binding domain (DBD), an intramolecular chaperone autoprocessing (ICA) domain, and a transmem- brane domain.48 Followed by a unique self-cleavage in the ICA domain, the DBD domain is liberated from the endoplasmic reticulum (ER) and translocated to the nucleus to regulate 49 FIGURE 3. Evolutionary conservation of amino acids affected by MYRF myelin gene expression. Through coexpression and genetic mutations. (A) p.Ser264fs; (B) p.R478P; and (C) p.R986X. interaction network analysis of MYRF, we reported shared genes among nanophthalmos and related diseases. Interesting- ly, coexpressed with MYRF, TMEM98, a known nanophthalmos gene, also encodes an ER-associated transmembrane protein.50

FIGURE 4. Gene coexpression and genetic interaction network analyses demonstrated that MYRF has correlations with validated pathogenic genes. MYRF was coexpressed with TMEM98 (nanophthalmos-related gene), RSPO1 (AL), BMP4 (MAC), and PITX3 (MAC). MYRF interacted with GLIS3 (angle-closure glaucoma).

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FIGURE 5. Pedigree charts of trios with nanophthalmos-relevant inherited-autosomal variants. The square represents male and circle represents female; black represents nanophthalmic patients and white represents unaffected parents. (A–I) Inherited-autosomal variants in each individual are labeled under each figure (square or circle).

TMEM98 binds to the C-terminal of MYRF and inhibits the self- the optic nerves in chronic angle-closure glaucoma, MYRF may cleavage of MYRF, functioning as a negative feedback regulator play a contributing role in the occurrence of chronic angle- of MYRF in OL differentiation and myelination.50 Conditional closure glaucoma. Both the Human Protein Atlas database and ablation of MYRF results in severe CNS dysmyelination, with Mouse ENCODE Project demonstrate that MYRF is highly stalling of oligodendrocytes at the premyelinating stage and expressed in stomach, intestine, lung, liver, and heart,52,53 exhibiting severe deficits in myelin gene expression.48 Emery supporting the role of MYRF beyond the CNS. Recent trio et al.51 have confirmed the lack of myelin ensheathment in the studieshavereportedDNMsinMYRF as candidates in optic nerves of conditional knockout mice by electron congenital diaphragmatic hernia and congenital heart dis- microscopy, whereas control optic nerves were actively ease.54,55 Most of them were damaging missense mutations myelinating. Considering the axonal and myelin damage in located in the conserved DBD and ICD. Additionally, MYRF was

TABLE 3. The Inherited Autosomal Variants in Nanophthalmic Patients

Patient Number Gene Mutation Frequency in ExAC ACMG Category ACMG Evidence

1 (Trio number 1) PRSS56 c.1066dupC, p.Gln356fs (het) 5/10538 Pathogenic PVS1, PM2, PP5, PP4 c.1687delC, p.Arg563fs (het) 0 Pathogenic PVS1, PM2, PM3, PP4 2 (Trio number 2) PRSS56 c.343dupG, p.Ala115fs (het) 0 Pathogenic PVS1, PM2, PP4 c.1508T>A, p.Met503Lys (het) 0 Likely pathogenic PM1, PM3, PP3, PP4 4 (Trio number 4) PRSS56 c.1066dupC, p.Gln356fs (het) 5/10538 Pathogenic PVS1, PM2, PP5, PP4 c.781G>A, p.Gly261Arg (het) 0 Likely pathogenic PM1, PM3, PP3, PP4 5 (Trio number 5) MFRP c.577_578delAG, p.Ser193fs (hom) 0 Pathogenic PVS1, PM2, PP4 6 (Trio number 6) PRSS56 c.1186G>A, p.Glu396Lys (het) 0 Likely pathogenic PM1, PM3, PP3, PP4 c.1183þ32_1186þ50del (het) 0 Uncertain significance PM1, PP4 7 (Trio number 7) PRSS56 c.1066dupC, p.Gln356fs (het) 5/10538 Pathogenic PVS1, PM2, PP5, PP4 c.1186G>A, p.Glu396Lys (het) 0 Likely pathogenic PM1, PM3, PP3, PP4 9 (Trio number 9) MFRP c.661C>T, p.Pro221Ser (het) 1/82594 Uncertain significance PM1, PP3, PP4 c.1411G>A, p.Val471Met (het) 3/120282 Uncertain significance PM1, PP3, PP4 11 (Trio number 11) MFRP c.498delC, p.Asn167fs (hom) 14/120550 Pathogenic PVS1, PM2, PP5, PP4

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FIGURE 6. The identified inherited autosomal variants in PRSS56 and MFRP.(A) Schematics of the functional domains of the PRSS56 protein. Tryp_SPc, Trypsin-like serine protease. (B) Schematics of the functional domains of the MFRP protein. p.Gln356fs and p.Asn167fs have been reported. CUB, cubilin-related domain; LDLa, low-density lipoprotein receptor class A domain; Fz, cysteine rich domain.

identified during the course of constructing a transcript map of population, further validation using an enlarged multicenter the region encompassing the BEST1 gene (OMIM 607854) and clinical collection of larger trios and sporadic patients and eye tissue-specific cDNA selection.56 BEST1 mutations have investigation of its molecular mechanisms are needed to been reported to be associated with autosomal-dominant ascertain the causative role of MYRF mutations in the vitreoretinochoroidopathy (ADVIRC), autosomal-recessive be- development of nanophthalmos. strophinopathy (ARB), and angle-closure glaucoma, all of In summary, trio-based WGS revealed the potential role of which have been reported to be associated with nano- DNMs in MYRF and rare inherited genetic variants in PRSS56 57,58 phthalmos. In our study, the two DNMs (c.789delC, and MFRP, which will likely help expand our understanding of p.S264fs and c.789dupC, p.S264fs) cause premature stop the mechanism of nanophthalmos. codons of MYRF, damage the normal protein structure and thus cause serious harm to the function of MYRF;the additional DNM (c.1433G>C, p.R478P) located in the DNA Acknowledgments binding domain of MYRF, leading to heterozygous arginine to The authors thank KingMed Diagnostics Group Co., Ltd. (Guangz- proline substitution at amino acid position 478 (Supplementa- hou, China) for their technical support in whole-genome ry Fig. S1); and the stop-gain mutation (c.2956C>T, p.R986X) sequencing; all the patients for their continued trust, confidence, led to the damage of Myelin gene regulatory factor-C-terminal and their willingness to participate in this research study which domain 2. The detailed locations of all mutations in MYRF are makes our findings possible; and express deep appreciation for the shown in Fig. 2E. The recurrence of MYRF mutations in trios mentors and colleague clinicians at the Zhongshan Ophthalmic and sporadic case supported the potential pathogenicity role Center, including Drs. Yizhi Liu, Minbin Yu, Yehong Zhuo, Mingkai for MYRF gene in Chinese patients with nanophthalmos. Lin, Xiulan Zhang, Jingjing Huang, and Chengguo Zuo, etc. Both PRSS56 and MFRP are known nanophthalmos genes. Supported by Major Project of National Natural Science Founda- A significant number of recessive nanophthalmos cases have tion of China (NSFC)-Guangdong Province Joint Fund (Grant been detected with mutations in PRSS56 and MFRP.11,12 Nair number: 3030902113080; Guangzhou, China), the Science and et al.59 reported that PRSS56Grm4 mutant mice had a shortened Technology Planning Project of Guangdong Province (Grant axial length and higher susceptibility to angle closure. MFRP number: 303090100502050-18; Guangzhou, China), Guangzhou seems to play an important role in ocular growth during Science and Technology Plan Project (Grant number: No. 2018- childhood, functioning as a regulator of ocular size.31 Our 1202-SF-0019; Guangzhou, China), Research Funds of the State Key results further confirmed the role of these two genes in the Laboratory of Ophthalmology (Grant numbers: pathogenesis of nanophthalmos. 30306020240020153, 30306020240020192, 3030902113058, Some limitations in this study should be considered. First, 3030902113118, PT1001022; Guangzhou, China), and Fundamen- there is no direct evidence supporting the causative connec- tal Research Funds of Sun Yat-sen University (Grant number: tion between MYRF and nanophthalmos, yet the recurrence of 16ykjc31; Guangzhou, China). MYRF mutations and overlapping coexpression and genetic Disclosure: C. Guo, None; Z. Zhao, None; D. Chen, None; S. He, interaction with confirmed nanophthalmos-candidate genes None; N. Sun, None; Z. Li, None; J. Liu, None; D. Zhang, None; indicate that MYRF is likely to be responsible for the J. Zhang, None; J. Li, None; M. Zhang, None; J. Ge, None; X. development of nanophthalmos. Second, the DNMs are Liu, None; X. Zhang, None; Z. Fan, None heterogeneous variants found in probands with nanophthal- mos, suggesting that their inheritance pattern is autosomal References dominant. However, we should be cautious about this conclusion, and more family data are needed to make a final 1. Nowilaty SR, Khan AO, Aldahmesh MA, Tabbara KF, Al-Amri A, conclusion. Third, because we only included the Chinese Alkuraya FS. Biometric and molecular characterization of

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