A Cross-Sectional Study in 731 Chinese Patients

Genetics Expanding the Phenotypic and Genotypic Landscape of Nonsyndromic High Myopia: A Cross-Sectional Study in 731 Chinese Patients

Xue-Bi Cai,1 Yi-Han Zheng,1 De-Fu Chen,1 Fang-Yue Zhou,1 Lu-Qi Xia,1 Xin-Ran Wen,1 Yi-Min Yuan,1 Fang Han,1 Shun-Yu Piao,2 Wenjuan Zhuang,2 Fan Lu,1 Jia Qu,1 A-Yong Yu,1 and Zi-Bing Jin1 1The Eye Hospital, School of Ophthalmology & Optometry, Wenzhou Medical University, National Center for International Research in Regenerative Medicine and Neurogenetics, National Clinical Research Center for Ophthalmology, State Key Laboratory of Ophthalmology, Optometry and Visual Science, Wenzhou, China 2Ningxia Medical University, People’s Hospital of Ningxia Hui Autonomous Region, Yinchuan, China

Correspondence: Zi-Bing Jin, The PURPOSE. High myopia (HM) is defined as a refractive error worse than 6.00 diopter (D). This Eye Hospital, Wenzhou Medical Uni- study aims to update the phenotypic and genotypic landscape of nonsyndromic HM and to versity, Wenzhou 325027, China; establish a biological link between the phenotypic traits and genetic deficiencies. [email protected]. A-Yong Yu, The Eye Hospital, Wenz- METHODS. A cross-sectional study involving 731 participants varying in refractive error, axial hou Medical University, Wenzhou length (AL), age, myopic retinopathy, and visual impairment. The phenotypic traits were 325027, China; analyzed by four ophthalmologists while mutational screening was performed in eight [email protected]. autosomal causative genes. Finally, we assessed the clinical relevance of identified mutations Jia Qu, The Eye Hospital, Wenzhou under the guidance of the American College of Medical Genetics and Genomics. Medical University, Wenzhou 325027 China; RESULTS. The relationship between refractive error and AL varied in four different age groups [email protected]. ranging from 3- to 85-years old. In adult groups older than 21 years, 1-mm increase in AL Submitted: July 5, 2019 conferred 10.84% higher risk of pathologic retinopathy (Category ‡2) as well as 7.35% higher Accepted: August 19, 2019 risk of low vision (best-corrected visual acuities <0.3) with P values < 0.001. The prevalence rates of pathologic retinopathy and low vision both showed a nonlinear positive correlation Citation: Cai X-B, Zheng Y-H, Chen D-F, with age. Forty-five patients were confirmed to harbor pathogenic mutations, including 20 et al. Expanding the phenotypic and genotypic landscape of nonsyndromic novel mutations. These mutations enriched the mutational pool of nonsyndromic HM to 1.5 high myopia: a cross-sectional study in times its previous size and enabled a statistically significant analysis of the genotype– 731 Chinese patients. Invest Oph- phenotype correlation. Finally, SLC39A5, CCDC111, BSG, and P4HA2 were more relevant to thalmol Vis Sci. 2019;60:4052–4062. eye elongation, while ZNF644, SCO2, and LEPREL1 appeared more relevant to refracting https://doi.org/10.1167/iovs.19-27921 media.

CONCLUSIONS. Our ﬁndings shed light on how multiple HM-related phenotypes are associated with each other and their link with gene variants. Keywords: high myopia, gene, mutation spectrum, genotype-phenotype correlation

yopia is deemed the leading cause of vision impairment in CCDC111 (encoding the PRIMPOL protein), LRPAP1, SLC39A5, M many countries.1–3 Patients with high myopia (HM; a LEPREL1, P4HA2, and BSG.18–32 Female-limited nonsyndromic refractive error worse than 6.00 diopter [D]) tend to exhibit HM has been associated with two X-linked genes, OPN1LW and excessive elongation of the eyeball and thinning of the sclera, ARR3. causing irreversible changes of the eye both functionally and It is challenging to establish a network connecting multiple morphologically.4,5 An international grading system of photo- clinical features or to reveal a biological link between the graphic classification for myopic maculopathy has been phenotypic traits and genetic deficiencies of HM. Importantly, developed.6 According to a World Health Organization report, large samples with a wide range of refractive errors, axial HM is producing a heavy burden on public health care and lengths (AL), ages, categories of myopic retinopathy, and best- socioeconomic well-being worldwide.7 corrected visual acuities (BCVA) are required for analyzing HM is a complex trait determined by both environmental either the phenotypic variation or the genetic predisposition in and genetic factors.8 To date, less time for outdoor activities, HM patients. In our study, we recruited 731 sporadic higher education, urban living environment, and off-axis individuals with nonsyndromic HM and comprehensively refraction have been identified etiologically for HM.9–13 sequenced eight known autosomal causative genes. Finally, Twenty-five chromosomal loci and over 100 gene variants have we successfully interpreted the relationships between different been reported for myopia by linkage analysis, genome-wide clinical characteristics and simultaneously identified 29 patho- association studies, and next-generation sequencing.14–17 Cur- genic mutations in 45 index cases, including 20 novel rently, eight autosomal genes have been discovered as mutations. In combination with previous reports, we further replicable disease-causing genes of nonsyndromic HM across revealed the genotype–phenotype correlation from a world- different study populations, including ZNF644, SCO2, wide perspective. To our knowledge, this is the largest cohort

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with HM that has been both phenotypically and genetically TABLE 1. Information on Refractive Error, Axial Length, and Age in the studied. 731 Patients With High Myopia Age, y Number of METHODS Subjects 3~21 22~40 41~59 ‡60

Study Subjects All 731 97 232 250 152 The study was in line with the tenets of the Declaration of SE, D Helsinki and approved by Ethics Committee of the Eye Hospital 6.00 to 9.99 164 37 75 36 16 of Wenzhou Medical University. A group of 731 Chinese 10.00 to 13.99 211 37 70 59 45 individuals diagnosed with nonsyndromic HM and 1082 14.00 to 17.99 136 12 31 59 34 18.00 to 21.99 128 8 31 51 38 healthy controls were recruited for this study. Informed 22.00 92 3 25 45 19 consent was obtained from each participant. The examinations AL, mm of refractive error and BCVA were carried out by objective 25.01–27.00 160 41 68 33 18 refractometer (Nidek, Aichi, Japan) and subjective refractom- 27.01–29.00 247 41 88 72 46 eter. Spherical equivalent refractive errors (SE) were calculated 29.01–31.00 168 10 39 70 49 as the total of the spherical power plus half of the cylindrical 31.01–33.00 110 5 29 50 26 power. Patients with hyperopia or severe astigmatism ( 1.00 < ‡33.01 46 0 8 25 13 D) were excluded from this study. AL measurements were completed using IOL-Master (Carl Zeiss Meditec AG, Jena, Germany). Patients with extreme HM and poor BCVA were Statistical Analysis further sent for and verified by retinoscopy optometry. Myopic fundus photos (CR-2 Retinal Camera; Canon, Saitama, Japan) One eye with higher myopic refractive error from per subject were graded by the same group of ophthalmologists as follows: was chosen for statistical analysis. However, when the eye has normal fundus (Category 0); tessellated fundus only (Category retinal detachment, in which case the measurement of AL 1); diffuse chorioretinal atrophy (Category 2); patchy chorio- would be inaccurate through IOL-Master, we would choose the retinal atrophy (Category 3); and macular atrophy (Category 4). other eye of the same subject. We performed multivariate ‘‘Plus’’ signs were defined as the presence of the following linear regression and multivariate logistic regression to additional features: (1) lacquer cracks, (2) choroidal neovascu- determine the association between (1) AL and SE; (2) myopic larization, and (3) Fuchs spot. Inconsistence of grading results retinopathy or visual impairment and AL or age; and (3) genetic by the ophthalmologists were addressed by further examina- findings and the clinical grading of AL and SE. The relationships tions of B-ultrasonography (AVISO; Quantel Medical, Cournon were depicted using the R language program (The R Project for d’Auvergne, France) and optical coherence tomography Statistical Computing, Vienna, Austria) and assessed with the (RTVue-100; Optovue, Fremont, CA, USA). v2 test. Two-tailed P values of < 0.05 were considered statistically significant. SPSS version 22.0 (IBM, Corp., Armonk, Comprehensive Mutational Screening NY, USA) was used for all statistical analyses. Peripheral blood sample was collected from each participant and genomic DNA was extracted. Pairs of primers specific for RESULTS coding sequences and intron/exon junctions of eight causative genes were designed. PCR was conducted and amplified Clinical Findings products were subjected to 1.5% agarose gel electrophoresis to confirm the fragment size, and subsequently sequenced. After The study cohort included 731 nonsyndromic HM patients direct Sanger sequencing, the results were analyzed and ranging from 3- to 85-years old. The value of SE ranged from compared with the University of California Santa Cruz Genome 6.00 to 38.00 D, while the value of AL ranged from 25.01 to Browser database. 36.73 mm. One eye with a higher myopic refractive error from each subject was chosen for further study of the phenotypic spectrum (Table 1). The fundus changes were recorded by the Pathogenicity Evaluation same group of ophthalmologists. Among them, 333 adult To evaluate the pathogenicity of candidate mutations, we patients older than 21 years were identified to have gradable explored the 1000 Genomes database (1000G), dbSNP135, the fundus photographs that were taken by the same panel of HapMap database, the Exome Aggregation Consortium (ExAC), ophthalmic technicians (Fig. 1). Typical ‘‘plus’’ signs and and in-house data. Sequence variants were standardized on the staphyloma are shown in Supplementary Figure S1. basis of Human Genome Variant Society variation nomencla- ture (in the public domain, http://www.hgvs.org/mutnomen/), Relationship Between Refractive Error and Axial checked by the Mutalyzer program (in the public domain, Length for Varying Ages http://www.lovd.nl/mutalyzer/), and assessed under the standards and guidelines of the American College of Medical A statistically significant trend of a 1.95 D (P < 0.001, R2 ¼ Genetics and Genomics (ACMG). Insertions or deletions 0.64) increase in refractive error per 1-mm growth in AL (DSE/ (InDels) were analyzed through the BWA (in the public DAL) was observed. In this specific cohort of 731 high-myopic domain, http://bio-bwa.sourceforge.net/) and GATK programs patients, 1-mm difference in AL equated with 2.21 D (P < (in the public domain, https://www.broadinstitute.org/gatk/). 0.001, R2 ¼ 0.65) of refractive error difference for the 3- to 21- Missense variants were evaluated with bioinformatics tools, year olds, 2.12 D (P < 0.001, R2 ¼ 0.67) for the 22- to 40-year and functional predictive scores were annotated through olds, 1.92 D (P < 0.001, R2 ¼ 0.60) for the 41- to 59-year olds, Wannovar (in the public domain, http://wannovar.wglab.org/). and 1.69 D (P < 0.001, R2 ¼ 0.53) for the group no younger Converted rank-scores were calculated using minimum–max- than 60 years (Fig. 2A). Linear regression adjusted for age imum normalization (Supplementary Text S1). showed that a 0.030 reduction (P < 0.001, R2 ¼ 0.98) in the

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FIGURE 1. Typical fundus photographs based on the international classiﬁcation and grading system for myopic maculopathy. Category 0 is characterized by no myopic retinal changes; Category 1 is characterized by tessellated fundus with easily visible outline of choroidal vessels around the arcade vessels; Category 2 is characterized by diffuse chorioretinal atrophy, showing a yellowish-white appearance of the posterior pole; Category 3 is characterized by patchy chorioretinal atrophy, which appears as well-deﬁned, grayish-white lesions around the centermost macular region; Category 4 is characterized macular atrophy, appearing as a widespread chorioretinal atrophic lesion affecting the foveal region.

FIGURE 2. The relationship between refractive error and AL for varying ages. (A) SE plotted as a function of AL for the whole high-myopic group as well as for each age group. The black line is the ﬁtted line for the whole group, while the blue line is for the 3- to 21-year olds; the purple line is for the 22- to 40-year olds; the reddish-orange line is for the 41- to 59-year olds; the green line is for the group older than 60 years. The length of the horizontal column in different colors represents the speciﬁc value of DSE/DAL for each age group. (B) SE:AL ratio plotted as a function of SE for different age groups. The SE:AL ratio decreases with increasing SE at distinct rates, ranging from 0.030/ to 0.027/1 D, for varying ages. (C) SE:AL ratio plotted as a function of AL for different age groups. The SE:AL ratio reduces fasted with increasing AL (0.059/mm) in the 3 to 21-year olds, followed by 0.055/mm in the 22- to 40-year olds, 0.046/mm in the 41- to 59-year olds, and 0.037/mm in the group older than 60 years.

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TABLE 2. Severity of Myopic Retinopathy and Visual Impairment in the 333 Adult Patients With Gradable Fundus Photographs

Grade of Myopic Maculopathy (International Photographic Classiﬁcation) BCVA Number of Subjects Category 0 Category 1 Category 2 Category 3 Category 4 V1 V2 V3 V4 V5

All 333 10 123 65 74 61 53 55 60 53 112 AL, mm 25.01–27.00 43 5 29 6 1 2 9 13 13 4 4 27.01–29.00 107 4 68 18 10 7 31 20 20 18 18 29.01–31.00 94 1 23 24 34 12 11 15 12 14 42 31.01–33.00 59 0 3 16 19 21 2 6 12 13 26 ‡33.01 30 0 0 1 10 19 013422 Age, y 22–35 52 4 30 10 6 2 21 17 5 3 6 36–49 83 6 37 16 17 7 22 17 14 18 12 50–63 111 0 41 18 23 29 8 17 22 15 49 64–77 81 0 13 20 26 22 1 4 17 17 42 ‡78 6 0 2 1 2 1 1020 3

SE:AL ratio was observed per 1-D increase in refractive error with Category 2 and 3 was much larger than that between for the 3- to 21-year olds, as well as a 0.029/1-D reduction (P those with Category 3 and 4 or between those with Category 4 < 0.001, R2 ¼ 0.98) for the 22- to 40-year olds, a 0.028/1-D and 5. Patients 50 years and older had a frequent presence reduction (P < 0.001, R2 ¼ 0.97) for the 41- to 59-year olds, and (142/198, 71.72%) of pathologic retinopathy as well as a high a 0.027/1-D reduction for the group no younger than 60 years risk (94/198, 47.47%) of low vision, while the other patients (P < 0.001, R2 ¼ 0.95) (Fig 2B). Additionally, we observed a younger than 50 years had a lower frequency (58/135, 42.96%) 0.059 reduction (P < 0.001, R2 ¼ 0.51) in the SE:AL ratio per 1- of pathologic retinopathy as well as a lower risk (18/135, mm increase in AL for the 3- to 21-year olds, as well as a 0.055/ 13.33%) of low vision. mm reduction (P < 0.001, R2 ¼ 0.54) for the 22- to 40-year 2 olds, a 0.046/mm reduction (P < 0.001, R ¼ 0.43) for the 41- Mutations Identified in the HM Cohort to 59-year olds, and a 0.037/mm reduction (P < 0.001, R2 ¼ 0.32) for the group older than 60 years (Fig. 2C). We comprehensively sequenced the 8 genes in 731 nonsyndromic HM patients and identified 29 pathogenic mutations Increasing Axial Length and Aging Confer Risks of in 45 index cases (Fig. 4A). Thereinto, 20 novel mutations had neither been reported previously nor been found in the 1000G Pathologic Retinopathy and Low Vision database (Table 3). We considered a variation as ‘‘pathogenic’’ Within the 333 patients with gradable fundus photographs, according to the following standards: (1) splice-site variations there was no statistically significant correlation between the fulfilled the GT-AT rules, (2) nonsense/frameshift variants were value of AL and age (P ¼ 0.59). The value of AL ranged from present, and (3) nonframeshift InDels/missense mutations 25.01 to 36.73 mm and was equally divided into 13 groups. To were predicted to be damaging or probably damaging by the enable comparison between all the AL groups, three groups standards and guidelines from ACMG and 11 bioinformatics with the longest AL but a small number of affected patients tools (Supplementary Table S1). Within the list, one was a were classified into one group, and so were the two groups nonsense mutation, and one was a frameshift mutation that with the shortest AL. The age was not normally distributed (P both severely affected protein function; one was a nonframe- < 0.01), and the Kruskal-Wallis H test confirmed that there was shift InDel, and 26 were missense mutations that were all no significant intergroup difference in age in the whole AL predicted to have deleterious effects on protein function group (P ¼ 0.96). In this cross-sectional study, the severity of through bioinformatics analyses (Fig 4B). All of these mutations macular retinopathy showed a positive correlation with the were absent from the control group, and their minor allele elongation of AL (Fig. 3A). Moreover, 1-mm increase in AL frequencies (MAF) were less than 0.05. conferred 10.84% higher risk of pathologic retinopathy (Category ‡2; P < 0.001). The detailed BCVAs of these Genetic Landscape and Genotype–Phenotype patients were classified into the following five stages: BCVA Correlations From the Worldwide Perspective ‡1.0 (V1); 0.7 BCVA < 1.0 (V2); 0.5 BCVA < 0.7 (V3); 0.3 BCVA < 0.5 (V4); and BCVA < 0.3 (V5) (Table 2). Likewise, We retrospectively summarized all the reported mutations in the visual impairment also showed a positive correlation with the eight genes from worldwide HM populations (Table 4). A the elongation of axial length (Fig. 3B). Notably, 1-mm increase total of 40 mutations have been reported in 103 individuals in AL conferred 7.35% higher risk of low vision (BCVA < 0.3; P from previous studies. In combination with our mutational < 0.001). findings, we achieved a clear picture of an expanding genetic Similarly, the prevalence rates of pathologic retinopathy and landscape of HM from a worldwide perspective (Fig. 5A). The low vision were calculated by age. The value of AL was not top three genes contributing to nonsyndromic HM were normally distributed in all age groups (P < 0.001), and the SLC39A5, ZNF644, and CCDC111. These three genes had a Kruskal-Wallis H test confirmed that there was no significant high frequency of mutations and should be given a priority intergroup difference in AL (P ¼ 0.26). The severity of macular during genetic screening of nonsyndromic HM patients. With retinopathy (Fig. 3C) and visual impairment (Fig. 3D) both the exception of SCO2, mutations in the other genes were showed a positive correlation with the growth of age. The age mostly reported in Asia-Pacific populations. Homozygous gap between those with Category 1 and 2 or between those mutations in LRPAP1 and LEPREL1 for autosomal-recessive

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FIGURE 3. The association between myopic retinopathy or visual impairment and AL or age. (A) The positive correlation between myopic retinopathy and AL. The bar graph on the left shows the number of patients with different categories of myopic retinopathy in different AL groups, with the gray dots representing the distribution of age; the proportional graph in the middle shows the percentage of patients with different categories of myopic retinopathy in different AL groups, with the gray line representing the mean age of each group; the boxplot on the right shows how patients with different categories of myopic retinopathy distribute over different ranges of AL. (B) The positive correlation between visual impairment and AL. The bar graph on the left shows the number of patients with different stages of visual impairment in different AL groups, with the gray dots representing the distribution of age; the proportional graph in the middle shows the percentage of patients with different stages of visual impairment in different AL groups, with the gray line representing the mean age of each group; the boxplot on the right shows how patients with different stages of visual impairment distribute over different ranges of AL. (C) The positive correlation between myopic retinopathy and age. The bar graph on the left shows the number of patients with different categories of myopic retinopathy in different age groups, with the gray dots representing the distribution of AL; the proportional graph in the middle shows the percentage of patients with different categories of myopic retinopathy in different age groups, with the gray line representing the mean AL of each group; the boxplot on the right shows how patients with different categories of myopic retinopathy are distributed over different age ranges. (D) The positive correlation between visual impairment and age. The bar graph on the left shows the number of patients with different stages of visual impairment in different age groups, with

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the gray dots representing the distribution of AL; the proportional graph in the middle shows the percentage of patients with different stages of visual impairment in different age groups, with the gray line representing the mean AL of each group; the boxplot on the right shows how patients with different stages of visual impairment distribute over different age ranges.

HM have been discovered as a result of consanguineous according to their ALs as follows: (1) Level 1: AL < 27 mm; (2) marriage, and mutations detected in LRPAP1 were all Level 2: 27 AL < 30 mm; (3) Level 3: AL ‡ 30 mm (extreme frameshift mutations, while those in other genes were mainly high axial myopia) (Fig 5B). According to the relationship missense. The only splicing mutation was discovered in BSG. between refractive error and AL for the general HM cohort in A total of 113 individuals harboring mutations in the eight our study, we categorized the refractive errors of all the genes, 68 from the previous studies and 45 from this study, patients into three grades corresponding to the levels of their were identified to have detailed records of refractive error, AL, ALs as follows: (1) Grade 1: SE > 11.00 D; (2) Grade 2: 17.00 and age. The eight individuals harboring LRPAP1 mutations < SE 11.00 D; (3) Grade 3: SE 17.00 D (Fig. 5C). The were all at young ages. In the other seven gene groups, age was patients with mutations in LRPAP1 showed severe abnormal- normally distributed (P ¼ 0.07), and one-way ANOVA further ities in refractive error and AL from an early age. The confirmed that there was no significant intergroup difference percentages of patients of different levels and grades were in age (P ¼ 0.70). All patients were categorized into three levels calculated for comparison in the other seven groups. Finally,

FIGURE 4. Mutational spectrum of nonsyndromic high myopia. (A) Pie chart showing mutations identiﬁed in this Chinese high-myopia cohort. Mutations marked with asterisks and in red font are novel mutations. The percentages of patients with mutations in different genes are shown in different colors. (B) Comparative pathogenic analysis of the 26 missense mutations. The detailed functional predictive rank-scores are shown in the heat map, with deeper colors indicating higher pathogenicity. The gray block represents the lack of predictive information. All predictions were made based on the standards and guidelines of the American College of Medical Genetics and Genomics and a series of bioinformatics tools. The variant in the bottom right (SCO2, p.R20P) is a previously reported nonpathogenic polymorphism that was used as a negative control.

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TABLE 3. Pathogenic Variants Identiﬁed in the Large High-Myopia Cohort of 731 Chinese Patients

Mutation Sample ID Gene Exon Nucleotide Protein Type Taster SIFT PolyPhen-2 EXAC 1000G SNP ID

WH172 SLC39A5 1 c.250C>T p.R84W Missense DC (0.747) D (0.004) PD (0.995) Rare Rare rs199681035 WH190 SLC39A5 1 c.250C>T p.R84W Missense DC (0.747) D (0.004) PD (0.995) Rare Rare rs199681035 S190 SLC39A5 1 c.178C>T p.R60W Missense N (0.999) D (0.003) PD (1.000) Rare Novel H392 SLC39A5 3 c.500A>G p.N167S Missense DC (0.999) T (0.673) PD (0.996) Novel Novel WH43 SLC39A5 7 c.1117C>T p.H373Y Missense DC (0.998) D (0.004) PD (0.998) Rare Novel WH232 SLC39A5 7 c.1117C>T p.H373Y Missense DC (0.998) D (0.004) PD (0.998) Rare Novel T135 SLC39A5 7 c.1121G>C p.S374T Missense DC (0.935) D (0.026) PD (0.997) Rare Rare rs74812296 HMS108 SLC39A5 7 c.1121G>C p.S374T Missense DC (0.935) D (0.026) PD (0.997) Rare Rare rs74812296 WH122 SLC39A5 7 c.1155G>A p.W385X Nonsense DC (1.000) – – Novel Novel H240 SLC39A5 9 c.1334T>C p.L445P Missense DC (0.999) T (0.080) PD (0.998) Novel Novel S291 SLC39A5 9 c.1327C>T p.R443W Missense DC (0.998) D (0.001) PD (0.998) Rare Rare rs140453919 T105 SLC39A5 10 c.1495C>T p.R499C Missense DC (0.999) D (0.001) PD (1.000) Rare Novel T118 SLC39A5 10 c.1580C>T p.T527I Missense DC (0.503) D (0.001) B (0.024) Novel Novel S20 CCDC111 2 c.265T>G p.Y89D Missense DC (0.999) D (0.009) PD (0.981) Rare Rare rs200857997 S218 CCDC111 3 c.389_390insG p.V131Gfs*6 Frameshift DC (1.000) Rare Rare rs558986236 T165 CCDC111 3 c.389_390insG p.V131Gfs*6 Frameshift DC (1.000) Rare Rare rs558986236 T182 CCDC111 3 c.389_390insG p.V131Gfs*6 Frameshift DC (1.000) Rare Rare rs558986236 S119 CCDC111 3 c.389_390insG p.V131Gfs*6 Frameshift DC (1.000) Rare Rare rs558986236 S152 CCDC111 3 c.389_390insG p.V131Gfs*6 Frameshift DC (1.000) Rare Rare rs558986236 M28 CCDC111 3 c.389_390insG p.V131Gfs*6 Frameshift DC (1.000) Rare Rare rs558986236 M57 CCDC111 3 c.389_390insG p.V131Gfs*6 Frameshift DC (1.000) Rare Rare rs558986236 M70 CCDC111 3 c.389_390insG p.V131Gfs*6 Frameshift DC (1.000) Rare Rare rs558986236 M156 CCDC111 3 c.389_390insG p.V131Gfs*6 Frameshift DC (1.000) Rare Rare rs558986236 WH175 ZNF644 2 c.1399A>G p.M467V Missense DC (0.999) D (0.006) PD (0.985) Rare Novel H205 ZNF644 2 c.2659G>C p.V887L Missense DC (0.973) D (0.000) PD (0.994) Rare Rare rs544944934 HMS90 ZNF644 2 c.2627A>G p.D876G Missense DC (0.998) D (0.001) P (0.457) Novel Novel H373 ZNF644 2 c.2608A>G p.T870A Missense DC (0.995) D (0.003) B (0.451) Rare Rare rs59922637 T116 ZNF644 3 c.3266A>G p.Y1089C Missense DC (0.999) D (0.000) PD (0.999) Rare Rare rs193167060 T149 ZNF644 3 c.3266A>G p.Y1089C Missense DC (0.999) D (0.000) PD (0.999) Rare Rare rs193167060 WH361 ZNF644 3 c.3266A>G p.Y1089C Missense DC (0.999) D (0.000) PD (0.999) Rare Rare rs193167060 H563 ZNF644 3 c.3261A>C p.E1087D Missense DC (0.856) D (0.007) B (0.001) Novel Novel HMS71 ZNF644 5 c.3966G>A p.M1322I Missense DC (0.998) D (0.002) P (0.924) Novel Novel HMS53 BSG 2 c.385C>T p.R129C Missense DC (0.999) D (0.001) PD (1.000) Rare Novel M118 BSG 2 c.385C>T p.R129C Missense DC (0.999) D (0.001) PD (1.000) Rare Novel WH289 BSG 2 c.385C>T p.R129C Missense DC (0.999) D (0.001) PD (1.000) Rare Novel WH86 BSG 2 c.70G>A p.G24S Missense DC (0.999) D (0.00) PD (1.000) Rare Rare rs539913003 M41 BSG 2 c.70G>A p.G24S Missense DC (0.999) D (0.00) PD (1.000) Rare Rare rs539913003 WH23 BSG 2 c.109G>A p.G37R Missense DC (0.999) D (0.007) PD (0.997) Novel Novel WH339 BSG 4 c.655G>A p.G219R Missense DC (0.832) D (0.003) PD (1.000) Novel Novel HM61 P4HA2 4 c.382G>A p.D128N Missense DC (0.999) D (0.000) PD (1.000) Novel Novel WH371 P4HA2 5 c.548_550delATC p.184delH Nonframeshift DC (0.999) Novel Novel InDel WH87 P4HA2 6 c.886G>T p.G296W Missense DC (0.999) D (0.000) PD (1.000) Rare Novel P111-1 SCO2 1 c.518A>T p.D173V Missense N (0.999) D (0.028) P (0.525) Novel Novel H360 SCO2 1 c.601G>C p.A201P Missense DC (0.716) D (0.012) PD (0.996) Novel Novel T111 SCO2 1 c.661A>G p.I221V Missense DC (0.999) D (0.030) B (0.003) Rare Novel DC, disease causing; D, deleterious; PD, probably damaging; P, possibly damaging; B, benign; N, polymorphism; T, tolerated. Rare, MAF < 0.05.

SLC39A5, CCDC111, BSG, and P4HA2 were found to be with considered gene function analyses to add to the list, statistically more relevant to ocular elongation, while ZNF644, further revealing the genotype–phenotype correlation. SCO2, and LEPREL1 seemed to function more on dioptric Through modeling by the Bennett-Rabbetts emmetropic media, which inﬂuences the measurement of the refractive schematic eye, an arbitrary value of DSE/DAL has been revealed error. In addition to the widely suspected role of these genes in (2.7 D/mm).33 Such numeric values of DSE/DAL have also ocular elongation, their potential role in the optical power of been evidenced by Deller et al.34 and Atchison et al.,4 who the dioptric media warrants further attention. cited 3.03 and 2.86 D/mm, respectively, for general populations. However, none of these studies focused speciﬁ- cally on high-myopic eyes, and how appropriate such values DISCUSSION are to the populations of varying ages is largely unknown. In this study, we exclusively examined HM participants and In the present study, a comprehensive phenotypic network of observed a value of 1.95 D/mm for the whole group. For the nonsyndromic HM is deciphered, within which the SE, AL, myopia population, 1-mm difference in AL would have a less grades of myopic retinopathy, and degrees of visual impairment profound effect on refractive error than for general popula- are positively correlated with each other to different extents in tions, which is supported by the longitudinal data from myopic different age groups. Moreover, genotyping of such a relatively children in a COMET study.35 Interage group comparisons sizable cohort of HM patients reveals additional gene variants were made in our work, and a decreasing value of DSE/DAL as

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TABLE 4. Summary of Known Mutations in the Eight Autosomal Causative Genes for Nonsyndromic High Myopia

Number of Affected Gene Exon Nucleotide Protein Type Ethnicity Individuals Reference

SLC39A5 1 c.250C>T p.R84W Missense Chinese 1 29 SLC39A5 1 c.141C>G p.Y47X Nonsense Chinese 6 27 SLC39A5 5 c.860C >T p.P287L Missense Chinese 1 29 SLC39A5 5 c.911T>C p.M304T Missense Chinese 2 27 SLC39A5 6 c.956G >C p.R319T Missense Chinese 1 29 SLC39A5 8 c.1238G>C p.G413A Missense Chinese 1 26 CCDC111 2 c.265T>G p.Y89D Missense Chinese 12 24 ZNF644 2 c.2014A>G p.S672G Missense Chinese 7 23, 26 ZNF644 2 c.2551G>C p.D851H Missense Chinese 1 26 ZNF644 2 c.2048G>C p.R683T Missense Chinese 1 26 ZNF644 2 c.1201A>G p.T401A Missense Chinese 1 32 ZNF644 2 c.2867C>G p.T956S Missense Chinese 1 32 ZNF644 2 c.2096G>A p.C699Y Missense Chinese 2 23 ZNF644 2 c.2038C>G p.R680G Missense Chinese 1 23 ZNF644 2 c.821A>T p.E274V Missense African American 1 31 ZNF644 2 c.725C>T p.T242M Missense American Caucasian 1 31 ZNF644 5 c.3833A>G p.E1278G Missense Chinese 1 32 LRPAP1 1 c.197delC p.Q67Sfs*8 Frameshift Chinese 1 26 LRPAP1 5 c.605delA p.N202Tfs*8 Frameshift Saudi 2 25 LRPAP1 7 c.863_864del p.I288Rfs*118 Frameshift Saudi 6 25 BSG 2 c.205C>T p.Q69X Nonsense Chinese 2 30 BSG 2 c.415þ1G>A - Splicing Chinese 1 30 BSG 5 c.661C>T p.P221S Missense Chinese 1 30 BSG 6 c.889G>A p.G297S Missense Chinese 1 30 LEPREL1 1 c.13C>T p.Q5X Nonsense Chinese 3 35 LEPREL1 1 c.292delC p.G100Afs*104 Frameshift Saudi Arabia 4 34 LEPREL1 5 c.1046T>C p.L349P Missense Chinese 1 30 LEPREL1 10 c.1523G>T p.G508V Missense Israeli-Bedouin 13 28 P4HA2 1 c.448A>G p.I150V Missense Chinese 1 36 P4HA2 4 c.419A>G p.Q140R Missense Chinese 2 36 P4HA2 6 c.871G>A p.E291K Missense Chinese 9 36 P4HA2 11 c.1327A>G p.K443X Nonsense Chinese 1 36 P4HA2 11 c.1349_1350delGT p.R451Gfs*8 Frameshift Chinese 1 36 SCO2 1 c.358C>T p.R120W Missense Chinese 1 26 SCO2 1 c.290C>T p.A97V Missense Japanese 1 33 SCO2 1 c.157C>T p.Q53X Nonsense European 7 22 SCO2 1 c.341G>A p.R114H Missense Middle Eastern 1 22 SCO2 1 c.418G>A p.E140K Missense European 1 22 SCO2 1 c.776C>T p.A259V Missense African American 1 22 SCO2 1 c.334C>T p.R112W Missense Chinese 1 26

age increased was observed, with R2 > 0.50 for each group. A the relationship between refractive error and AL for varying previous study based on UK populations showed a similar ages and found an obviously decreasing slope of the reduction finding of a decreasing value of DSE/DAL along with an as age increased. Thus, we assumed that Cruickshank et al.36 increasing age, but with lower coefficients of determination obtained an approximate value of 0.05/mm reduction for all between mean SE and AL (6- to 7-year olds: R2 ¼ 0.21; 12- to 13- age groups due to the narrow age range in their study. year-olds: R2 ¼ 0.24; and 18- to 25-year olds: R2 ¼ 0.45). Consequently, the wide age ranges in our study would help Notably, a composite graph of data for all three groups better understand the relationship between refractive error exhibited a better correlation.36 Thus, we speculated that the and AL. The older eye could not be considered to have the age range was the reason for the differences in correlation same mathematic relationship as a younger eye, probably due between different studies, with a larger range giving a higher to the fluidity in the coordination of ocular components at correlation coefficient. different stages of eye development. For instance, the aging Optical calculations based on Gullstrand reduced model eye crystalline lens has been shown to undergo a flattening of parameters predict a reduction in the SE:AL ratio as the AL curvatures, a decrease in the refractive index and other increases.37 We discovered a 0.059 reduction in SE:AL ratio per substantial age-related changes,38–41 which may partially 1-mm increase in AL for the 3- to 21-year olds, 0.055 for the 22- account for the nonlinear and nonconstant change in the to 40-year olds, 0.046 for the 41- to 59-year olds, and 0.037 for value of refractive error during the ocular elongation. the group no younger than 60 years. However, Cruickshank et AL increases and aging are significant risk factors for myopic al.36 observed a 0.05 reduction per 1-mm increase in AL for all retinopathy.42 Though continued progression of retinopathy age groups. We then carefully analyzed the plotted figures and elongation of AL with time have been demonstrated in showing a reduction of 0.05/mm and the equations reflecting multiple studies, how the severity of myopic retinopathy is

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FIGURE 5. Genetic landscape of highly myopic eyes and comparison of phenotypic severities between patients with mutations in different genes. (A) Heat maps showing the genetic landscape in worldwide high-myopia populations. The heat map on the left uses different colors to show the number of patients with different mutations. Mutations marked with # are discovered in our study, while those additionally marked with asterisks are novel mutations. The heat map on the right uses different colors to show the patients with different mutation types. (B) The percentages of patients with different levels of AL. The grayish line represents level 1: AL < 27 mm; the dark gray line represents level 2: 27 AL < 30 mm; the dark line represents level 3: AL ‡ 30 mm (extreme high axial myopia). (C) The percentages of patients with different grades of refractive error. The grayish line represents grade 1: SE > 11.00 D; the dark gray line represents grade 2: 17.00 D < SE 11.00 D; the dark line represents grade 3: SE 17.00 D.

relevant to the varying AL remains unclear.43–45 The wide included 298 nonsyndromic HM patients, which only identified range of AL and the similar distribution of age in different AL seven causative mutations in ZNF644, SCO2, LRPAP1, and groups in our study allowed a reliable cross-sectional study on SLC39A5, with no mutations discovered in LEPREL1.22 Other the association between the category of myopic retinopathy studies only focused on one or two genes in a rather limited and the value of AL. To have such an explicit understanding of number of patients. In this work, we comprehensively the implications of a progressively elongating AL to the visual sequenced the eight causative genes in a cohort of 731 HM system avoids obscuring the true nature of the myopic change patients. Mutations discovered in this cohort were analyzed by and helps correctly estimate the efficacy of myopia control bioinformatics tools after confirming their low frequencies in interventions. The age gaps between different categories authoritative databases and absence in the geographically showed that it would be a long-term process for the young matched control group. Ten mutations in SLC39A5 were populations with HM to develop retinopathy and they might identified in 13 index cases, including seven novel mutations. A well retain good vision before the pathologic changes loss-of-function in SLC39A5 interrupts the well-known path- occurred. Once the retinopathy was triggered, however, it way TGF-b/bone morphogenic protein, which regulates sclera would progress quickly in the following years. metabolism through modulation of the extracellular matrix, The important role of genetic factors in the occurrence of and is thus assumed to be associated with HM.23 Two HM has been implicated in several studies since the first effort CCDC111 mutations were identified in 10 index cases, was made to decipher the hereditary determinants in the including one missense mutation (Y89D) previously reported 1960s.46 The largest cohort that has been genetically studied in a four-generation Chinese family.20 Further study established

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that the major DNA replication defect relevant to this mutant Yuan, None; F. Han, None; S.-Y. Piao, None; W. Zhuang, None; was likely to contribute, at the molecular and cellular levels, to F. Lu, None; J. Qu, None; A.-Y. Yu, None; Z.-B. Jin, None the onset of HM.47 Another CCDC111 mutation (V131Gfs*6)is only present in Asian populations, indicating that it is probably Reference a specific mutation for HM in Asian. Seven mutations in ZNF644 were identified in nine index cases, including four 1. Kempen JH, Mitchell P, Lee KE, et al.; for the Eye Diseases novel mutations. ZNF644 has been recognized as a zinc finger Prevalence Research Group. The prevalence of refractive transcription factor widely expressed and is involved in eye errors among adults in the United States, Western Europe, and development, potentially contributing to axial elongation.27 Australia. Arch Ophthalmol. 2004;122:495–505. Four BSG mutations were identified in seven cases, including 2. Sperduto RD, Seigel D, Roberts J, et al. Prevalence of myopia three novel mutations. We discovered BSG as a causative gene in the United States. Arch Ophthalmol. 1983;101:405–407. for the elongation of AL and the occurrence of early-onset 3. Lin LL, Chen CJ, Hung PT, et al. Nation-wide survey of myopia HM.26 Three novel mutations in P4HA2, encoding prolyl 4- among schoolchildren in Taiwan, 1986. Acta Ophthalmol hydroxylase 2, were identified in three index cases. Mutations Suppl. 1988;185:29–33. of P4HA2 were supposed to result in unstable collagens in the 4. Atchison DA, Jones CE, Schmid KL, et al. Eye shape in sclera, and the disrupted sclera were responsible for the longer emmetropia and myopia. Invest Ophthalmol Vis Sci. 2004;45: AL of the eye as well as other HM phenotypes.32 Three novel 3380–3386. mutations in SCO2 were identified in three index cases. SCO2 5. Rada JA, Shelton S, Norton TT. The sclera and myopia. Exp encodes a copper homeostasis protein whose deficiencies Eye Res. 2006;82:185–200. have been linked to photoreceptor loss and myopia with 6. Ohno-Matsui K, Kawasaki R, Jonas JB, et al. International increased scleral wall elasticity.18 By adding these 20 novel photographic classification and grading system for myopic mutations, we enriched the mutational pool of nonsyndromic maculopathy. Am J Ophthalmol. 2015;159:877–883. HM to 1.5 times its previous size. 7. Bourne RR, Stevens GA, White RA, et al. Causes of vision loss The eight genes were all hypothesized to play roles in worldwide, 1990-2010: a systematic analysis. Lancet Glob scleral remodeling and ocular elongation. Although AL is Health. 2013;1:e339–e349. important biologically, refractive status is clinically a complex 8. Pan, CW, Ramamurthy D, Saw SM. Worldwide prevalence and variable determined by the optical power of the cornea and the risk factors for myopia. Ophthalmic Physiol Opt. 2012;32:3– lens in addition to the AL.5 Whether the mutations in these 16. genes influence the function of the dioptric media within the 9. Sherwin JC, Reacher MH, Keogh, RH, et al. The association eye has rarely been investigated. We here supposed SLC39A5, between time spent outdoors and myopia in children and CCDC111, BSG, and P4HA2 were more relevant to ocular adolescents: a systematic review and meta-analysis. Ophthal- elongation, while LEPREL1, ZNF644, and SCO2 had a greater mology. 2012;119:2141–2151. impact on the dioptric media. The first SCO2 study for HM 10. Morgan I, Rose K. How genetic is school myopia? Prog Retin successfully induced myopia in a mouse model by applying a Eye Res. 2005;24:1–38. 15.00-D lens over one eye and detected a significant decrease 11. Guo Y, Liu LJ, Xu L, et al. Outdoor activity and myopia among in messenger RNA levels of SCO2,18 suggesting that SCO2 was primary students in rural and urban regions of Beijing. closely relevant to refractive conditions. Visual experience has Ophthalmology. 2013;120:277–283. been relevant to human myopia, because children with corneal 12. Lu B, Congdon N, Liu X, et al. Associations between near opacities or eyelid ptosis can develop myopia.48 Patients with work, outdoor activity, and myopia among adolescent mutations in LEPREL1 were found to have variable expressivity students in rural China: the Xichang Pediatric Refractive of early-onset cataracts,31 which might partially account for the Error Study report no. 2. Arch Ophthalmol. 2009;127:769– development of HM. ZNF644 was supposed to follow the 775. trend of other transcription factor genes pathogenic to ocular 13. He M, Xiang F, Zeng Y, et al. Effect of time spent outdoors at diseases and play an important role in both dioptric media and school on the development of myopia among children in china: the ocular wall. Further studies are needed to elucidate the a randomized clinical trial. JAMA. 2015;314:1142–1148. exact mechanisms of these genes in eye development. 14. Young TL, Metlapally R, Shay AE. Complex trait genetics of In conclusion, our findings shed light on a network refractive error. Arch Ophthalmol. 2007;125:38–48. connecting multiple clinical features of HM, updating the 15. Wojciechowski R. Nature and nurture: the complex genetics current knowledge of how these features are associated with of myopia and refractive error. Clin Genet. 2011;79:301–320. each other. Moreover, by adding 20 novel mutations to the 40 16. Zhang, Q. Genetics of Refraction and Myopia. Prog Mol Biol previously reported mutations from the worldwide perspec- Transl Sci. 2015;134:269–279. tive, we were able to expand the genetic landscape of 17. Hysi PG, Wojciechowski R, Rahi JS, et al. Genome-wide nonsyndromic HM and further establish a biological link association studies of refractive error and myopia, lessons between the phenotypic traits and genetic deficiency. learned, and implications for the future. Invest Ophthalmol Vis Sci. 2014;55:3344–3351. Acknowledgments 18. Tran-Viet KN, Powell C, Barathi VA, et al. Mutations in SCO2 are associated with autosomal-dominant high-grade myopia. The authors thank Kai-Jing Zhou, MD, PhD, for the assistance of Am J Hum Genet. 2013;92:820–826. clinical data collection. 19. Shi Y, Li Y, Zhang D, et al. Exome sequencing identifies Supported by grants from National Key Research and Development ZNF644 mutations in high myopia. PLoS Genet.2011;7: Program of China (2017YFA0105300; Beijing, China), National e1002084. Natural Science Foundation of China (81970838; Beijing, China), 20. Zhao F, Wu J, Xue A, et al. Exome sequencing reveals CCDC111 Zhejiang Provincial Natural Science Foundation of China mutation associated with high myopia. Hum Genet. 2013;132: (LQ17H120005; Zhejiang, China), and the Ministry of Education 913–921. 111 project (D16011; Beijing, China). 21. Aldahmesh MA, Khan AO, Alkuraya H, et al. Mutations in Disclosure: X.-B. Cai, None; Y.-H. Zheng, None; D.-F. Chen, LRPAP1 are associated with severe myopia in humans. Am J None; F.-Y. Zhou, None; L.-Q. Xia, None; X.-R. Wen, None; Y.-M. Hum Genet. 2013;93:313–320.

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