Genetic Spectrum and Genotype-Phenotype Correlations in a Chinese Cohort With Nanophthalmos With Secondary Angle-Closure Glaucoma

Abstract

Purpose

The purpose of this study was to explore the genetic and clinical features of nanophthalmos with secondary angle-closure glaucoma (NSACG) in a Chinese cohort. This was a prospective cross-sectional study of 157 eyes from 88 Chinese patients with NSACG.

Methods

The participants underwent ocular and systemic examinations and whole-exome sequencing. The main outcome measures were pathogenic genetic variants, axial length (AL), refractive spherical equivalent (SE), vitreous chamber depth (VCD), white-to-white (WTW), radius of corneal curvature (flat and steep K: K1 and K2), anterior chamber depth (ACD), lens vault (LV), lens thickness (LT), extent of angle closure, anterior segment crowding value, retinal nerve fiber layer (RNFL) thickness, central subfield thickness (CST) in macular, cup-to-disc ratio (C/D), mean defect in visual field, and onset age of angle-closure glaucoma (ACG).

Results

Seventy-eight variants (51.14%) were identified in 45 patients, including 20 in PRSS56 (44.44%) and 14 in MFRP (31.11%) with autosomal recessive (AR) inheritance, 8 in MYRF (17.78%), and 3 in TMEM98 (6.6%) with autosomal dominant (AD) inheritance. Individuals with genetic diagnosis were associated with shorter AL, higher SE, larger K1 and K2, shallower ACD, greater angle closure extent, larger LT/AL, shorter VCD, and higher incidence of retinal detachment. Compared with AR cases, patients with AD showed younger ACG onset, longer AL, lower SE, smaller K1 and K2, longer VCD, thinner CST of the macula, and more severe visual field defects.

Conclusions

Among Chinese patients with NSACG, PRSS56 and MFRP were the predominant AR variants, whereas MYRF and TMEM98 were the main AD variants. Genetic diagnosis exhibited shorter AL and a more crowded anterior segment, leading to accelerated glaucoma progression. The faster glaucoma progression in AD cases highlights the need for early intervention.

Nanophthalmos, an uncommon disorder characterized by diminutive yet structurally normal ocular anatomy, arises from embryonic developmental arrest during early embryogenesis.¹ Defined by ocular dimensions at least 2 standard deviations below the age-adjusted mean (i.e. <21 mm), nanophthalmos manifests with pronounced hyperopia, increased retinochoroidal sclera thickness (RCS; >1.7 mm), diminutive corneal diameter, shallow anterior chamber, venous plexus anomalies, and absence of additional ocular anomalies.^2,3 These distinctive anatomic traits predispose individuals to complications such as angle-closure glaucoma (ACG), ciliary zonule defects, uveal effusion syndrome (UES), retinal folds and detachment, strabismus, and amblyopia, thereby significantly impairing vision.⁴

Considered a disorder of normal ocular growth stemming in the early stages of embryogenesis, nanophthalmos exhibits a robust genetic underpinning. Previous investigations have implicated 6 genes directly associated with nanophthalmos: serine protease 56 (PRSS56, OMIM 613858),⁵ membrane-type frizzled-related protein (MFRP, OMIM 606227),⁶ myelin regulatory factor (MYRF, OMIM 608329),⁷ transmembrane protein 98 (TMEM98, OMIM 615949),⁸ crumbs homolog 1 (CRB1, OMIM 604210),⁹ and bestrophin 1 (BEST1, OMIM 607854).¹⁰ PRSS56, MFRP, and CRB1 exhibit an autosomal recessive (AR) inheritance pattern, MYRF and TMEM98 are characterized by autosomal dominant (AD) inheritance, whereas BEST1 demonstrates both AR and AD inheritance patterns. Predominantly, variants in PRSS56 and MFRP elucidate the majority of cases,⁴ with rare occurrences in other genes. However, discrepancies exist regarding the fraction of nanophthalmos cases attributable to variants in known genes with the rate of genetic diagnosis ranging from 18.8% to 69.2%, potentially influenced by varying study inclusion criteria and sampled populations across different studies.^11–13 Our previous trio-based whole-genome sequencing study for nanophthalmos has revealed that MYRF coding variants explained part of the nanophthalmos trios and sporadic cases, as well as inherited genetic variants in PRSS56 and MFRP.¹⁴ Whereas the genetic spectrum and its demographics has not been thoroughly investigated in China yet.

Additionally, genotype-phenotype correlations in patients with nanophthalmos remain partially elucidated due to limited sample sizes. Recently, a study involving 67 patients with nanophthalmos from Chinese families evaluated the differences in clinical features of nanophthalmos with mutations in PRSS56, MFRP, MYRF, and TMEM98 and assessed the association between ACG and nanophthalmos resulting from these variants.¹⁵ However, they exclusively compared clinical features caused by these different genes without contrasting them with cases lacking gene variants. Moreover, the clinical characteristics of their patients were influenced by complications such as ACG and UES, potentially introducing bias in assessing the relationship between specific genetic etiologies and ocular phenotypes. In our clinic, nanophthalmos with secondary ACG (NSACG) is the primary reason for patients seeking medical attention, and it represents the most difficult condition to treat. Thus, in this study, we focus on patients with NSACG, and extended the investigation to an expanded cohort of Chinese families utilizing whole-exome sequencing, aiming to discern the underlying genetic etiology and clinical manifestations of NSACG.

Methods

Subjects

In this prospective study, 88 patients with NSACG were consecutively recruited at Beijing Tongren Hospital from March 2021 to March 2024. The diagnosis of NSACG was based on the following criteria: (1) characters of nanophthalmos including axial length (AL) less than 21 mm and without morphologic malformation^16,17; (2) in combination with secondary ACG not related to other reasons, such as trauma, uveitis, neovascularization, or increased episcleral venous pressure¹⁸; and (3) without severe ocular malformation, multiple non-ophthalmic syndromic features. Patients with apparent secondary UES observed during fundus examination, which could have altered the intraocular morphology, were excluded from this study. Ethical approval was obtained from the Ethic Committee of Beijing Tongren Hospital and was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from the parents or legal guardians of children younger than 18 years old. The geographic distribution of all subjects was recorded based on their places of birth and classified into northern and southern China, as well as seven major geographic regions (Fig. 1).¹⁹

Figure 1.

Geographic distribution of patients with NSACG. The analysis of the geographic distribution was focused on 74 remaining patients. The division of the North and the South is the Qinling Mountain-Huaihe River Line. Northeast China includes Heilongjiang Province, Jilin Province, and Liaoning Province; North China includes Beijing, Tianjin, Hebei Province, Shanxi Province, and Inner Mongolia Autonomous Region; East China includes Shanghai, Jiangsu Province, Zhejiang Province, Anhui Province, Jiangxi Province, Shandong Province, Fujian Province, and Taiwan; South China includes Guangdong Province, Guangxi Province, Hainan Province, Hong Kong, and Macao. Central China includes Henan Province, Hunan Province, and Hubei Province; Northwest China includes Shanxi Province, Gansu Province, Qinghai Province, Ningxia Hui Autonomous Region, and Xinjiang Uygur Autonomous Region. Southwest China includes Yunnan Province, Guizhou Province, Sichuan Province, and Tibet Autonomous Region.

Clinical Examination

All eligible individuals underwent the standard ophthalmic clinical evaluations, including best corrected visual acuity (BCVA), slit-lamp examination, intraocular pressure (IOP) measurement (Goldmann applanation tonometry), and fundus examination. Angle closure extent was quantified in degrees, with 90 degrees assigned for each closed quadrant on gonioscopy. Manifest refraction was performed to determine the refractive spherical equivalent (SE). The age at examination, as well as the estimated onset age of ACG based on the history of ocular hypertension and glaucomatous optic neuropathy was recorded. The AL, the horizontal corneal diameter (white-to-white [WTW]), corneal curvature (K), including K1 (flattest keratometry value) and K2 (steepest keratometry value), and vitreous chamber depth (VCD) were obtained with the IOL Master 700 (software version 5.4; Carl Zeiss Meditec, Inc., Dublin, CA, USA). Central corneal thickness (CCT), anterior chamber depth (ACD), lens vault (LV), and lens thickness (LT) were measured with the swept-source anterior segment optical coherence tomography (AS-OCT; CASIA2 system, Tomey, Nagoya, Japan). The cup-to-disc ratio (C/D) was assessed by two experienced ophthalmologists (authors X.Y. and Y.S.) via direct ophthalmoscopy. In cases of significant discrepancy, a third ophthalmologist (author Z.F.) adjudicated the final judgment. The retinal nerve fiber layer (RNFL) thickness, central subfield thickness (CST) of the macula, and thickness of the ganglion cell layer and inner plexiform layer (GCL + IPL) were measured by Cirrus high-definition OCT (HD-OCT; Carl Zeiss Meditec, Dublin, CA, USA). Foveal hypoplasia (FH) defined as a flatter or less distinct foveal contour, absence of the foveal avascular zone, and persistence of inner retinal layers,¹⁵ retinal detachment (RD) defined as the neurosensory retina separated from the underlying retinal pigment epithelium (RPE),²⁰ and retinoschisis defined as a split between the RNFL and GCL,²¹ were evaluated by Cirrus HD-OCT (Fig. 2). Retinal vascular tortuosity, characterized by the abnormal twisting or coiling of retinal blood vessels, was evaluated using scanning laser ophthalmoscope (SLO) imaging (P200T, Optos Daytona, UK; see Fig. 2).²² The visual field was measured by Humphrey perimetry (Carl Zeiss Meditec, Dublin, CA, USA) with 24–2 Swedish interactive threshold algorithm (SITA) fast strategy. The anterior segment crowding value was calculated as (LT-ACD)/AL.²³ Ciliochoroidal detachment was evaluated using ultrasound biomicroscopy (UBM; model SW-3200L; Tianjin Suowei Electronic Technology Co., Ltd., Tianjin, China) and was defined as a hypoechoic space between the sclera and the detached ciliary body and choroid (see Fig. 2).²⁴ Images used to evaluate these features that were fractured, distorted, or blurred were not included in the analysis. Two blinded observers (authors X.Y. and H.Z.) judged and categorized the images independently, and if there was a discrepancy between the two observers, a third expert (author Y.S.) made the judgment.

Figure 2.

Typical clinical imaging features of patients with nanophthalmos with genetic diagnosis. (A) Foveal hypoplasia (yellow triangle) of a patient with compound heterozygous variants of PRSS56 in c.1066dup (p.Gln356ProfsTer152) and c.1186G>A (p.Glu396Lys) demonstrated by Cirrus HD-OCT. (B) Retinal detachment (red triangle) of a patient with heterozygous variant of TMEM98 in c.602G>C (p.R201P) demonstrated by Cirrus HD-OCT. (C) Retinoschisis (blue triangle) of a patient with homozygous variants of MFRP in c.58G>T (p.Glu20Ter) demonstrated by Cirrus HD-OCT. (D) Retinal vascular tortuosity (red arrowheads) of the same patient in B demonstrated by scanning laser ophthalmoscope imaging (SLO, P200T; Optos Daytona, UK). (E) Ciliochoroidal detachment (yellow arrowheads) of the same patient in C evaluated using ultrasound biomicroscopy (UBM, model SW-3200L; Tianjin Suowei Electronic Technology Co., Ltd., Tianjin, China).

Whole Exome Sequencing and Variant Analysis

Whole exome sequencing was performed as previously described.²⁵ Briefly, genomic DNA was extracted from peripheral blood and the coding DNA was enriched using the SureSelect Human All Exon Kit (Agilent V6, Santa Clara, CA, USA). Paired-end sequencing was performed with the Novaseq 6000 sequencer (Illumina, San Diego, CA, USA) with an average sequencing depth of 102.53X. Alignment and variant calling were performed according to the Genome Analysis Toolkit (GATK) and the Variant Call Format (VCF) files were then analyzed using ANNOVAR software. The variants were filtered using the following criteria: (1) low-quality variants were removed by GATK recommended filters; (2) variants present in the public genetic variant databases, Genome Aggregation Database (GnomAD version 2.0.1), with an allele frequency < 2% were included; and (3) nonsense variants, frameshift variants, splice site, or predicted damaging missense variants by Combined Annotation Dependent Depletion (CADD), Sorting Intolerant From Tolerant (SIFT), PolyPhen2, or VariantTaster were included. Additionally, the American College of Medical Genetics and Genomics (ACMG) guidelines were used. The sequencing depth in subjects in the discovery stage ranged from 99.17 to 259.84 and the genome coverage ranged from 99.30% to 99.88%.

Statistical Analysis

Quantitative data with normal distribution were presented as mean ± standard deviation (SD), whereas those of abnormal distribution were expressed as median and quartiles. Differences between the two groups were analyzed by Student's t-test or the Mann-Whitney U test as appropriate. Kruskal-Wallis tests were used for multiple group comparisons, followed by Bonferroni-corrected post hoc tests when significant differences were detected. Qualitative data were compared by the chi-square test. In shorter eyes, there was a tendency toward a thicker RNFL with a decrease in AL.^26,27 Therefore, only C/D measured by direct ophthalmoscopy and mean defect (MD) in the visual field result was used as the main indicators of the degree of optic nerve damage. Generalized estimating equations (GEEs) were conducted to assess the influence of AD and AR on C/D and visual field defects, with results reported using the Wald test. Logistic regression was used to adjust for the effects of age and AL on the incidence of retinal detachment, retinoschisis, and ciliochoroidal detachment. P < 0.05 was considered statistically significant. Data analysis were performed using SPSS (SPSS Inc., Chicago, IL, USA).

Results

Clinical Phenotyping of Patients With NSACG

We included 88 Chinese patients with NSACG (157 eyes) from 87 unrelated non-consanguineous families, with a mean age at examination of 44.5 ± 15.0 years old and an estimated mean onset age of ACG at 42.24 ± 15.46 years old (Table 1). Detailed clinical characteristics for each patient were provided in Supplementary Table S1. Thirty-one (35.6%) patients were men and 57 (64.4%) patients were women. The mean BCVA (Log MAR) was 0.97  ±  0.83, and the mean SE was 7.82  ±  4.56 diopters (D), and all patients had a history of elevated IOP, with a mean maximum IOP of 32.68 ± 14.70 millimeters of mercury (mm Hg). Using the IOL Master 700, the mean AL, WTW, and VCD were 18.25 ± 2.06 mm, 11.29 ± 0.54 mm, and 11.68 ± 2.17 mm, respectively. The mean K1 and K2 were 46.74 ± 2.77 D and 48.25 ± 2.97 D, respectively. Anterior segment parameters measured by AS-OCT showed a mean CCT of 537.33 ± 65.19 µm, ACD of 1.89 ± 0.56 mm, LV of 1.34 ± 0.52 mm, and LT of 4.69 ± 0.90 mm. The mean degree of angle closure and mean anterior segment crowding value was 298.28 ± 80.11 degrees and 0.201 ± 0.057, and the mean MD in the visual field was −17.17 ± 11.68 decibels (dB). Other ocular parameters included a mean C/D of 0.66 ± 0.26, a mean RNFL thickness of 91.16 ± 36.00 µm, a mean GCL + IPL thickness of 67.39 ± 22.94 µm, and a mean CST of 286.15 ± 102.11 µm, as measured by Cirrus HD-OCT. Foveal hypoplasia was observed in 39 eyes (49.4%), retinal detachment in 15 eyes (19.0%), retinoschisis in 30 eyes (38.0%), retinal vascular tortuosity in 36 eyes (45.6%), and ciliochoroidal detachment in 23 eyes (29.1%).

Table 1.

Clinical Characteristics of 88 Chinese Patients With NSACG (157 Eyes) and the Comparison Between Patients With or Without Genetic Diagnosis

	Total	Negative	Genetic Diagnosis	P Value
Age at examination, y	44.52 ± 14.98	49.80 ± 14.25	39.57 ± 13.98	<0.001***
Estimated onset age, y	42.24 ± 15.46	48.47 ± 14.40	36.33 ± 14.12	<0.001***
Gender, M:F	31:57	12:31	19:26	0.160
BCVA, Log MAR	0.97 ± 0.83	0.64 ± 0.0.67	1.30 ± 0.85	<0.001***
SE, D	7.82 ± 4.56	4.81 ± 3.40	10.66 ± 3.31	<0.001***
Maximum IOP, mm Hg	32.68 ± 14.70	31.08 ± 13.91	34.23 ± 15.44	0.317
AL, mm	18.25 ± 2.06	19.96 ± 1.09	16.64 ± 1.32	<0.001***
K1, D	46.74 ± 2.77	45.48 ± 2.51	48.31 ± 2.24	<0.001***
K2, D	48.25 ± 2.97	47.07 ± 2.85	49.72 ± 2.44	<0.001***
VCD, mm	11.68 ± 2.17	12.97 ± 1.66	10.09 ± 1.60	<0.001***
WTW, mm	11.29 ± 0.54	11.32 ± 0.58	11.27 ± 0.50	0.685
ACD, mm	1.89 ± 0.56	1.90 ± 0.56	1.69 ± 0.48	0.049*
CCT, µm	537.33 ± 65.19	544.32 ± 68.83	528.90 ± 60.31	0.277
LT, mm	4.69 ± 0.90	4.71 ± 1.01	4.68 ± 0.79	0.817
LV, mm	1.34 ± 0.52	1.30 ± 0.41	1.38 ± 0.62	0.523
Extent of angle closure, degrees	298.28 ± 80.11	281.92 ± 85.04	326.25 ± 64.00	0.009**
ACD/AL	0.101 ± 0.033	0.095 ± 0.028	0.102 ± 0.030	0.202
LT/AL	0.26 ± 0.06	0.24 ± 0.05	0.27 ± 0.06	0.018*
VCD/AL	0.62 ± 0.068	0.64 ± 0.064	0.60 ± 0.06	0.002**
Anterior segment crowding value	0.17 ± 0.057	0.15 ± 0.052	0.19 ± 0.057	0.002**
C/D	0.66 ± 0.26	0.58 ± 0.23	0.74 ± 0.27	<0.001***
Thickness of RNFL, µm	89.87 ± 36.27	79.19 ± 29.36	101.40 ± 39.93	0.026*
Thickness of GCL + IPL, µm	67.37 ± 23.72	65.52 ± 25.26	69.29 ± 22.38	0.583
CST of macula, µm	286.15 ± 102.11	235.75 ± 106.38	336.54 ± 68.47	<0.001***
Visual field defect, MD	−17.17 ± 11.68	−12.34 ± 10.66	−21.63 ± 10.83	<0.001***
Foveal hypoplasia, n, %	39, 49.4	18, 46.2	21, 52.5	0.573
Retinal detachment, n, %	15, 19.0	1, 2.6	14, 35.0	<0.001***
Retinoschisis, n, %	30, 38.0	17, 43.6	13, 32.5	0.031*
Retinal vascular tortuosity, n, %	36, 45.6	15, 38.5	21, 52.5	0.949
Ciliochoroidal detachment, n, %	23, 29.1	5, 12.8	18, 45.0	0.002**

ACD, anterior chamber depth; AD, autosomal dominant; AL, axial length; AR, autosomal recessive; BCVA, best corrected visual acuity; CCT, central corneal thickness; C/D, cup/disc ratio; CST, central subfield thickness; GCL + IPL, ganglion cell layer and inner plexiform layer; IOP, intraocular pressure; K, corneal curvature; LT, lens thickness; LV, lens vault; RNFL, retinal nerve fiber layer;SE, refractive spherical equivalent; WTW, white-to-white.

^*P < 0.05.

^**P < 0.01.

^***P < 0.001.

Genotyping of Patients With NSACG

A definite genetic diagnosis by whole-exome sequencing was achieved in 45 out of 88 patients (51.14%). Among the 45 patients, a total of 78 variants were identified (37 frameshift, 30 missense, 5 nonsense, 5 splice site, and 1 intron deletion; see Supplementary Table S2), of which 4 patients with PRSS56 variants, 3 patients with MFRP variants, and 4 patients with MYRF variants have been reported in our previous study.¹⁴ These variants were confirmed by Sanger sequencing and were further validated by segregation analysis. The genetic etiologies found in this cohort were as follows: PRSS56 (n = 20, 22.73%), MFRP (n = 14, 15.91%), MYRF (n = 8, 9.09%), and TMEM98 (n = 3, 3.41%; Fig. 3). Among the 20 patients carrying variants of PRSS56, 7 (35.00%) were homozygote and 13 (65.00%) were compound heterozygote. Of all the 40 variants, 26 (65.00%) could be classified as frameshift variants, followed by 12 (30.00%) missense variants, with 1 (2.50%) splicing variant and 1 (2.50%) intron deletion. The comparison with Lev Prasov's¹² and Owen M. Siggs’¹³ studies was shown in Figure 4A. Further, 23 variants (57.50%) were in exon 9, of which 19 (47.50%) were the identified variant c.1066dup, followed by 3 (7.50%) c.1186G>A variants, as well as 1 c.1182_1183del variant (Fig. 4B).

Figure 3.

Distribution of gene variants. The pie chart represented the distribution of each gene variants in all of the 88 patients with NSACG.

Figure 4.

Genetic features of patients with NSACG with PRSS56, MFRP, MYRF, and TMEM98 variants. (A) The outer ring, middle ring, and inner ring stand for the variant types of PRSS56 in Owen M Siggs’ study, Lev Prasov’s study, and our study, separately. (B) Schematic of variants and loci identified for PRSS56. Each white circle represents one variant or loci. (C) The outer ring, middle ring, and inner ring stand for the variant types of MFRP in Owen M Siggs’s study, Lev Prasov’s study, and our study, separately. (D) Schematic of variants and loci identified for MFRP. Each white circle represents one variant or loci. (E) Schematic of variants and loci identified of MYRF. (F) Schematic of variants and loci identified of TMEM98. Each white circle represents one variant or loci.

Among the 14 patients with variants of MFRP, 7 (50%) were homozygote and 7 (50%) were compound heterozygote. Of the total 28 variants, 13 (46.43%) missense variants predominated, followed by 6 frameshift variants, 4 nonsense variants, 3 splicing variants, and 2 codon variants. Figure 4C illustrates distribution of mutational types comparing to the Lev Prasov's¹² and the Owen M. Siggs’¹³ studies. The greatest number of variants was found in exon 12, which contained 6 missense changes: 4 c.1486G>A, 1 c.1411G>A, and 1 c.1435_1437delinsCAA (Fig. 4D). For patients with homozygous or compound heterozygous, the variants were identified as originating from each parent, indicating that the variants appear in trans.

All patients with MYRF variants were heterozygote, with three frameshift variants, two missense variants, two nonsense variants, and one splicing variant (Fig. 4E). The discovery of three DNMs of MYRF (c.789del, p.S264fs, c.789dup, p.S264fs, and c.1433G>C, p.R478P) and a stop-gain MYRF variant (c.2956C>T, p.R986X) has been demonstrated in our previous study.¹⁴ Apart from that, a novel splicing variant c.1115+5G>A was found in intron 7, a novel missense variant c.1468C>A was found in exon 10, and a novel frameshift variant c.3195-20_3263delinsTGGAGA was found in exon 25. Whereas in the Owen M. Siggs’ study and the Lev Prasov’s study, only 1 frameshift variant c.3361del in exon 26 was reported.^12,13

All the 3 patients carrying variants of TMEM98 were heterozygote, of which 2 had missense variants and 1 had frameshift variant (Fig. 4F). In the Owen M. Siggs’ and the Lev Prasov’s studies, the reported TMEM98 variants are all missense variants.

Geographic Distribution of Patients With NSACG and Their Gene Variants

Excluding 14 patients due to the missing information, we focused our analysis on the geographic distribution of the remaining 74 patients. We observed that 39 patients hailed from southern China, whereas 35 patients were from the northern regions. The positive rate of genetic diagnosis did not exhibit a statistically significant difference between the southern and northern regions (59.0% vs. 45.7%, P = 0.254). However, a significant difference in the positive rates of genetic diagnosis among the different regional groups was detected by further zoning of China into 7 major geographic regions²⁸: 6 (100%) in 6 patients from Southwest China, 13 (68.4%) in 19 patients from East China, 2 (50.0%) in 4 patients from Northwest China, 5 (35.7%) in 14 patients from North China, 2 (33.33%) in 6 patients from Northeast China, 8 (72.73%) in 11 from Central China, and 3 (21.4%) in 14 patients from South China (P = 0.006; see Fig. 1). There was no significant difference between the AD and the AR group in the distribution of the southern and northern regions (P = 0.355). Especially, we found that the AD gene variants were more common in the Central China Plain (CCP)²⁹ than in other regions compared with the AR gene variants (P = 0.032).

Genotype-Phenotype Correlations in Patients With NSACG

The clinical characters of patients with or without genetic diagnosis were summarized in Table 1. Compared with patients without genetic diagnosis, those with genetic diagnosis showed a younger onset age of ACG, worse BCVA, and higher SE with shorter AL, larger K1 and K2, shorter VCD, shallower ACD, greater angle closure extent and lower VCD/AL, bigger LT/AL, and higher anterior segment crowding value, as well as bigger C/D, thicker average thickness of RNFL and CST of the macula, and more severe visual field defects (all P < 0.05). They also showed higher incidence of retinal detachment, retinoschisis, and ciliochoroidal detachment (all P < 0.05). The maximum IOP, LT, LV, WTW, CCT, ACD/AL, thickness of GCL + IPL and incidence of foveal hypoplasia, retinal degeneration, and retinal vascular tortuosity were not significantly different between patients with and without genetic diagnosis (all P > 0.05). We further compared clinical features across four genotypes (Table 2) and almost all the comparisons between the PRSS56 group and the MFRP group showed no significant difference (P1 > 0.05), as well as comparisons between the MYRF group and the TMEM98 group (P6 > 0.05). Patients with variants of PRSS56 and MFRP were then grouped into the AR group (n = 34), whereas those with variants of MYRF and TMEM98 were grouped into the AD group (n = 11). In general, compared with the AR group, patients with AD variants showed a younger estimated onset age of ACG, with longer AL, smaller K1 and K2, longer WTW, longer VCD, smaller LT/AL and anterior segment crowding value, and thinner CST of macular (all P < 0.05). There were no significant differences in other parameters between patients with AD and AR variants (all P > 0.05; Table 3). After adjusting for the influence of age at examination and AL via GEE or logistic regression, the analysis revealed that individuals with genetic diagnosis had thicker CST (Wald χ² = 12.172, P < 0.001), higher incidence of retinal detachment (Wald χ² = 25.579, P < 0.001), and ciliochoroidal detachment (Wald χ² = 12.910, P < 0.001) compared with those without variants, and patients in the AD group showed larger C/D (Wald χ² = 4.173, P = 0.041) and more severe visual field defects than the AR group (Wald χ² = 5.756, P = 0.016).

Table 2.

Comparison of Clinical Data Among Patients With Variants in PRSS56, MFRP, MYRF and TMEM98

	PRSS56 (n = 20)	MFRP (n = 14)	MYRF (n = 8)	TMEM98 (n = 3)	P	P1	P2	P3	P4	P5	P6
Age at examination, y	43.24 (34.75 to 49.00)	40.00 (23.00 to 61.00)	35.00 (27.00 to 35.00)	34.33 (29.00 to 40.00)	0.114	—	—	—	—	—	—
Estimated onset age, y	41.65 (33.00 to 48.00)	37.17 (22.25 to 53.50)	28.13 (17.00 to 34.00)	30.33 (29.00 to 32.00)	0.006**	0.327	<0.001***	0.010*	0.183	0.603	0.389
Gender, M:F	10:10	5:9	1:7	3:0	0.904	—	—	—	—	—	—
BCVA, Log MAR	1.23 (0.70 to 1.40)	1.54 (0.70 to 2.30)	0.76 (0.30 to 1.00)	1.38 (0.43 to 2.42)	0.021	0.147	0.011*	0.788	0.006**	0.694	0.209
SE, D	12.60 (10.88 to 14.62)	10.66 (9.50 to 13.00)	7.78 (7.00 to 10.00)	8.58 (6.75 to 10.25)	<0.001***	0.019*	<0.001***	0.002**	0.001**	0.024*	0.876
Maximum IOP, mm Hg	29.99 (16.00 to 40.00)	32.41 (15.75 to 44.00)	30.37 (15.70 to 38.00)	33.00 (17.25 to 55.00)	0.885	—	—	—	—	—	—
AL, mm	15.93 (15.48 to 16.43)	16.16 (15.47 to 16.88)	18.11 (17.48 to 18.27)	18.61 (17.43 to 19.35)	<0.001	0.266	<0.001***	<0.001***	<0.001***	<0.001***	0.311
K1, D	49.15 (48.13 to 49.72)	49.00 (47.88 to 50.16)	45.52 (44.38 to 46.80)	46.67 (46.13 to 47.34)	<0.001	0.682	<0.001***	0.001**	0.001**	0.026*	0.167
K2, D	50.29 (49.02 to 51.69)	50.36 (48.82 to 52.03)	46.52 (45.29 to 47.14)	47.72 (47.56 to 47.90)	<0.001	0.413	<0.001***	<0.001***	0.001**	0.019*	0.019
VCD, mm	10.64 (10.02 to 11.15)	10.93 (1.19 to 11.59)	12.59 (12.04 to 13.08)	13.11 (12.13 to 14.11)	<0.001	0.457	0.003**	0.005**	0.003**	0.005**	0.462
WTW, mm	11.22 (10.81 to 11.60)	11.05 (10.70 to 11.33)	11.43 (10.80 to 12.12)	12.07 (12.00 to 12.20)	0.031	0.256	0.544	0.006**	0.254	0.009**	0.241
ACD, mm	1.65 (1.44 to 1.91)	1.75 (1.43 to 1.98)	2.23 (1.94 to 2.37)	2.07 (1.68 to 2.82)	0.011	0.604	0.003**	0.149	0.007**	0.205	0.303
CCT, µm	531.28 (498.25 to 551.25)	498.93 (479.00 to 518.50)	553.80 (468.50 to 653.00)	540.50 (540.00 to 541.50)	0.047	0.029*	0.517	0.632	0.459	0.011*	0.135
LT, mm	4.90 (4.62 to 5.19)	4.51 (4.28, 4.80)	4.80 (4.43, 5.29)	4.52 (4.12, 4.79)	0.253	—	—	—	—	—	—
LV, mm	1.41 (1.18 to 1.80)	1.46 (1.18 to 1.70)	1.66 (1.10 to 1.71)	1.14 (0.93 to 1.38)	0.328	—	—	—	—	—	—
Extent of angle closure, degrees	299.12 (180.00 to 360.00)	326.25 (292.50 to 360.00)	324.00 (270.00 to 360.00)	330.00 (270.00 to 360.00)	0.576	—	—	—	—	—	—
ACD/AL	0.103 (0.087 to 0.115)	0.107 (0.088 to 0.114)	0.122 (0.108 to 0.133)	0.111 (0.087 to 0.148)	0.184	—	—	—	—	—	—
LT/AL	0.29 (0.28 to 0.32)	0.28 (0.27 to 0.31)	0.26 (0.24 to 0.27)	0.24 (0.22 to 0.27)	<0.001	0.069	0.002**	0.005**	0.033*	0.022*	0.269
VCD/AL	0.66 (0.64 to 0.68)	0.67 (0.66 to 0.69)	0.70 (0.68 to 0.72)	0.71 (0.70 to 0.73)	0.012	0.343	0.027*	0.013*	0.043*	0.024*	0.806
Anterior segment crowding value	0.20 (0.17 to 0.23)	0.19 (0.16 to 0.21)	0.14 (0.11 to 0.17)	0.13 (0.11 to 0.17)	<0.001	0.398	0.002**	0.008**	0.004**	0.017*	0.841
C/D	0.71 (0.50 to 1.00)	0.82 (0.72 to 1.00)	0.75 (0.40 to 0.90)	0.78 (0.45 to 1.00)	0.469	—	—	—	—	—	—
Thickness of RNFL, µm	121.00 (95.00 to 57.00)	88.12 (60.25 to 99.75)	103.25 (84.00 to 120.25)	86.50 (57.50 to 104.00)	0.197	—	—	—	—	—	—
Thickness of GCL + IPL, µm	81.75 (65.25 to 97.75)	58.71 (45.00 to 69.00)	66.75 (48.50 to 87.75)	74.50 (56.50 to 99.00)	0.171	—	—	—	—	—	—
CST of macula, µm	370.14 (311.00 to 465.00)	359.62 (313.25 to 426.75)	274.50 (267.25 to 282.25)	261.50 (248.25 to 271.25)	0.008	0.862	0.008**	0.008**	0.042**	0.042*	0.083
Visual field defect, MD	−19.86 (−30.50 to −9.48)	−24.31 (−32.00 to −15.95)	−20.88 (−30.00 to −6.20)	−21.90 (−32.75 to −11.77)	0.446	—	—	—	—	—	—
Foveal hypoplasia, n, %	9, 69.23	4, 44.44	2, 50.00	1, 50.00	1.000	—	—	—	—	—	—
Retinal detachment, n, %	5, 38.46	3, 33.33	0, 0	1, 50.00	1.000	—	—	—	—	—	—
Retinoschisis, n, %	6, 46.15	3, 33.33	2, 50.00	2, 100	1.000	—	—	—	—	—	—
Retinal vascular tortuosity, n, %	7, 53.85	4, 44.44	3, 75.00	1, 50	1.000	—	—	—	—	—	—
Ciliochoroidal detachment, n, %	6, 46.15	5, 55.56	0, 0	2, 100	1.000	—	—	—	—	—	—

ACD, anterior chamber depth; AD, autosomal dominant; AL, axial length; AR, autosomal recessive; BCVA, best corrected visual acuity; CCT, central corneal thickness; C/D, cup/disc ratio; CST, central subfield thickness; GCL + IPL, ganglion cell layer and inner plexiform layer; IOP, intraocular pressure; K, corneal curvature; LT, lens thickness; LV, lens vault; RNFL, retinal nerve fiber layer; SE, refractive spherical equivalent; WTW, white-to-white.

P1, comparison between PRSS56 and MFRP.

P2, between PRSS56 and MYRF.

P3, between PRSS56 and TMEM98.

P4, between MFRP and MYRF.

P5, between MFRP and TMEM98.

P6, between MYRF and TMEM98.

^* P < 0.05.

^** P < 0.01.

^*** P < 0.001.

Table 3.

Comparison of Clinical Data Between the AD Group and the AR Group

	AD	AR	P Value
Age at examination, y	34.00 (29.00 to 37.50)	40.00 (30.00 to 52.25)	0.028*
Estimated onset age, y	29.00 (25.00 to 32.00)	40.00 (29.50 to 51.50)	0.001**
Gender, M:F	4:7	15:19	0.736
BCVA, Log MAR	0.70 (0.30 to 1.91)	1.30 (0.70 to 2.00)	0.086
SE, D	10.66 (9.50 to 13.00)	12.60 (10.88 to 14.63)	0.017*
Maximum IOP, mm Hg	35.00 (18.35 to 51.00)	38.50 (16.00 to 48.50)	0.754
AL, mm	17.76 (17.46 to 18.46)	16.16 (15.49 to 16.68)	<0.001***
K1, D	46.13 (44.53 to 47.20)	49.38 (48.58 to 50.29)	<0.001***
K2, D	47.30 (45.74 to 47.68)	50.78 (49.66 to 51.98)	<0.001***
VCD, mm	10.85 (9.89 to 12.03)	9.49 (8.66 to 9.92)	0.004**
WTW, mm	12.00 (11.50 to 12.20)	11.05 (10.76 to 11.53)	<0.001**
ACD, mm	1.90 (1.82 to 2.14)	1.61 (1.42 to 1.90)	0.084
CCT, µm	540.00 (499.50 to 541.00)	513.00 (483.00 to 539.75)	0.302
LT, mm	4.79 (4.45 to 5.09)	4.72 (4.53 to 5.09)	0.835
LV, mm	1.46 (1.10 to 1.71)	1.23 (1.04 to 1.49)	0.300
Extent of angle closure, degrees	326.25 (292.50 to 360)	299.12 (180 to 360)	0.163
ACD/AL	0.108 (0.097 to 0.119)	0.102 (0.087 to 0.114)	0.350
LT/AL	0.259 (0.243 to 0.278)	0.29 (0.27 to 0.32)	0.002**
VCD/AL	0.60 (0.56 to 0.62)	0.59 (0.55 to 0.60)	0.415
Anterior segment crowding value	0.16 (0.14 to 0.19)	0.20 (0.18 to 0.22)	0.023*
C/D	0.77 (0.45 to 1.00)	0.74 (0.50 to 1.00)	0.653
Thickness of RNFL, µm	94.00 (63.00 to 104.75)	114.50 (81.00 to 150.00)	0.159
Thickness of GCL + IPL, µm	64.50 (56.00 to 71.50)	63.00 (53.00 to 94.00)	0.605
CST of macula, µm	276.50 (267.25 to 295.00)	335.00 (311.00 to 396.00)	0.010*
Visual field defect, MD	−21.61 (−31.00 to −9.58)	−20.97 (−32.00 to −10.23)	0.821
Foveal hypoplasia, n, %	5, 50.0	16, 53.3	0.855
Retinal detachment, n, %	3, 30.0	11, 36.7	1.000
Retinoschisis, n, %	3, 30.0	10, 33.3	1.000
Retinal vascular tortuosity, n, %	5, 50.0	16, 53.3	1.000
Ciliochoroidal detachment, n, %	3, 30.0	15, 50	0.464

ACD, anterior chamber depth; AD, autosomal dominant; AL, axial length; AR, autosomal recessive; BCVA, best corrected visual acuity; CCT, central corneal thickness; C/D, cup/disc ratio; CST, central subfield thickness; GCL + IPL, ganglion cell layer and inner plexiform layer; IOP, intraocular pressure; K, corneal curvature; LT, lens thickness; LV, lens vault; RNFL, retinal nerve fiber layer; SE, refractive spherical equivalent; WTW, white-to-white.

^* P < 0.05.

^** P < 0.01.

^*** P < 0.001.

Discussion

In the current study, we broadened the array of coding variants within PRSS56, MFRP, MYRF, and TMEM98 among a large cohort of Chinese individuals diagnosed with NSACG and 45 (51.14%) of 88 patients were detected with gene variants. The distribution of variants were as follows: 20 (22.73%) in PRSS56, 14 (15.91%) in MFRP, 8 (9.09%) in MYRF, and 3 (3.41%) in TMEM98. Among a total of 78 variants in 45 patients, we have found 2 novel frameshift variants, 2 novel missense variants, and 1 novel splicing variant in PRSS56; 4 novel missense variants, 3 novel splicing variants, 2 codon variants, and 2 nonsense variants in MFRP; 3 heterozygous novel variants including 1 splicing variant, 1 missense variant, and 1 frameshift variant in MYRF, and 1 novel frameshift variant in TMEM98. Together, this study represents a thorough assessment of rare genetic variants in NSACG and the genotype-phenotype correlations, with a particular emphasis on their relationship with the progression of ACG.

Comparatively, the relative contribution of genes to the pathogenesis of nanophthalmos was significantly different among our and prior reports. Studies from the United States and Norway reported higher incidence of PRSS56 and MFRP variants among cohorts with nanophthalmos and high hyperopia. In the American cohort, the incidence of PRSS56 and MFRP variants was 7.5% and 9.4% in 53 families, with a criterion of AL < 21 mm.¹² Similarly, a Chinese study focusing on high hyperopia reported PRSS56 and MFRP variant rates of 8% and 6% in 46 patients, with a criterion of bilateral refraction errors of at least +5.00 D.³⁰ Conversely, in the Norwegian study, the positive rates for PRSS56 and MFRP were 40% and 8%, respectively, in 25 patients, with AL < 20 mm as the inclusion criterion.¹¹ Particularly, previous studies reported little variants of TMEM98 and MYRF. Our data for the 88 patients with nanophthalmos within the Chinese population suggested a relative higher positive rate of genetic diagnosis, which may be attributed to several distinctions from other described cohorts. First, when we use stricter ACMG criteria and upon excluding variants classified as “uncertain” by ACMG, the total positive rate decreased to 29.54% (Table 4). Second, our cohort was collected primarily from the glaucoma department of Beijing Tongren Hospital, one of the most famous ophthalmic centers in China, and our cohort included patients with nanophthalmos who were all diagnosed with secondary ACG. Third, when considering stringent AL cutoffs, the positive rate increased to 70.31% (45/64) when AL < 20 mm, and 95.34% (41/43) when AL < 18 mm, which aligns with our observation that shorter AL correlates with a higher incidence of gene variants.

Table 4.

Positive Rates of Different Genetic Diagnose in Different Criteria

	PRSS56, n, %	MFRP, n, %	MYRF, n, %	TMEM98, n, %	Total, n, %
AL < 21 mm, n = 88	20, 22.7	14, 15.9	8, 9.1	3, 3.4	45, 51.1
AL < 20 mm, n = 64	20, 31.2	14, 21.9	8, 12.5	3, 4.7	45, 70.3
AL < 18 mm, n = 43	20, 46.5	14, 32.6	6, 14.0	1, 2.3	41, 95.4
AL < 15 mm, n = 6	4, 66.7	2, 33.3	0, 0	0, 0	6, 100
Stricter ACMG, n = 88 criteria	12, 13.6	6, 6.8	6, 6.8	1, 1.3	25, 28.4

ACMG, American College of Medical Genetics and Genomics; AL, axial length.

Table 4 shows the positive rates of PRSS56, MFRP, MYRF, and TMEM98 in different AL diagnostic criteria and in stricter ACMG criteria (exclude those with “uncertain” variants) in 88 patients with NSACG.

Despite these factors, we were still able to demonstrate the presence of variant diversity across different populations by analyzing the geographic distribution of patients with NSACG and their gene variants in our study. In this study, we find that the AD gene variants were more common in the CCP than in other regions compared with the AR gene variants (P = 0.032). These differences are possibly related to the economic and cultural constraints.³¹

In this study, a notably shorter AL, higher SE, larger K1 and K2, shallower ACD, greater extent of angle closure, larger LT/AL, higher anterior segment crowding value, shorter VCD, and lower VCD/AL were found among individuals with genetic diagnosis compared with those without. This suggests that the arrest of AL elongation may be more pronounced in genetically predisposed individuals. Previous studies suggested that the characteristics of nanophthalmos were the proportional reduction in size of ACD, VCD, and AL, with normal-size lens.³² Our finding of similar WTW and ACD/AL but a lower VCD/AL in individuals with a genetic diagnosis, compared with those without, suggests that these genes may have an additional influence on vitreous chamber expansion³³ (Fig. 5). Moreover, the increased occurrence of retinal detachment, retinoschisis, and ciliochoroidal detachment in individuals with a genetic diagnosis could be attributed to dysfunction in fluid transfer through the retina, choroid, and sclera, driven by a shorter AL, subsequently thicker sclera, and higher pressure in the vortex vein.³⁴ However, the higher incidence of RD and ciliochoroidal detachment, even after adjusting for age and AL, may indicate potential mechanical traction exerted by the underdeveloped vitreous—likely due to these variants.³⁵ Although further investigation is warranted, these features also indicate a potential increased susceptibility to uveal expansion in these eyes, as fluid outflow from the choroid is suppressed by thick sclera and elevated vortex vein pressure.³⁶ Additionally, although the ACD/AL was similar between groups, individuals with a genetic diagnosis showed shorter AL and a shallower ACD with a similarly sized lens, resulting in a larger LT/AL ratio and higher anterior segment crowding value. The higher anterior segment crowding, along with the increased susceptibility of the uvea to expansion in shorter eyes of individuals with a genetic diagnosis, may predispose them to an earlier onset of ACG and more severe optic nerve damage.^37,38

Figure 5.

The simple diagram of AL in patients with NSACG with various genetic basis. (A) Normal eyeball. (B) Patients with NSACG without a genetic diagnosis. (C) Patients with AD variants. (D) Patients with AR variants. Patients with NSACG in different groups showed similar LV, LT, ACD/AL, and VCD/AL. Patients with AR variants have more extreme ocular parameters including shorter AL, larger K1 and K2, shorter WTW, shorter VCD, larger LT/AL, and higher anterior segment crowding value. ACD, anterior chamber depth; AL, axial length; LT, lens thickness; LV, lens vault; VCD, vitreous chamber depth; WTW, white to white. Patients with NSACG also demonstrate thickening of the retina, choroid, and sclera. However, these findings are not depicted in the simplified diagram as detailed measurements of these parameters were not included in this study.

Patients with PRSS56 variants had clinical features that closely paralleled those observed in individuals with MFRP variants. Likewise, the phenotypic characteristics associated with MYRF variants resembled those seen in patients with TMEM98 variants. Collectively, individuals in the AR group demonstrated more pronounced ocular abnormalities—including shorter AL, reduced VCD, and greater LT/AL—resulting in a higher degree of anterior segment crowding than those in the AD group. The AR group also had larger K1 and K2 values and a shorter WTW, indicating limited corneal expansion. These differences may arise from the varying pathogenic effects of different genes on ocular development. Recent studies suggest that PRSS56 and MFRP may regulate prenatal AL growth,^4,39,40 which aligns with our finding that AL in the AR group (16.16 mm) is shorter than the typical AL at birth (17.34 ± 0.55 mm).⁴⁰ Other studies suggested that PRSS56 and MFRP regulated ocular growth during both prenatal and postnatal stages, thus affecting the anterior segment and leading to more pronounced ocular parameters.^31,32 On the contrary, the effect of TMEM98 on eye enlargement mainly occurred during postnatal development, and it was confined to the posterior retina, with the anterior segment unaffected.^2,41,42 Meanwhile, previous studies reported that both TMEM98 and MYRF are endoplasmic reticulum (ER)-associated transmembrane proteins.⁴³ TMEM98 interacts with MYRF, suppressing self-cleavage and nuclear localization, which may lead to similar clinical features in affected patients.^41,43 Although the molecular mechanisms behind different gene effects on ocular development were warranted to be further studied, the AD group in this study had earlier glaucoma onset and more severe visual field defects compared to the AR group, even after adjusting for age and AL. The incidence of retinal detachment, retinoschisis, and ciliochoroidal detachment did not differ significantly between genotypes, either with or without adjustment for age and AL. These findings suggest that anterior segment crowding and uvea expansion may not be the primary causes of faster ACG progression in nanophthalmos with different gene variants. MYRF is a key transcriptional regulator essential for oligodendrocyte differentiation and central nervous system (CNS) myelination.⁴⁴ As the retina is part of the CNS, individuals with TMEM98 and MYRF variants may experience primary optic nerve damage and be more vulnerable to elevated IOP. Although the exact mechanism requires further study, our findings highlight the need for intensive clinical care for patients with NSACG with these variants.

Our study encountered several limitations. First, the onset age of the majority of patients was estimated based on examination age and disease duration, introducing a degree of subjectivity. Second, due to many patients presenting at advanced disease stages or having received prior treatment elsewhere, there may be statistical errors in the clinical parameters. Hence, a multicenter, longitudinal study with larger sample sizes is imperative to elucidate genotype-phenotype relationships and prognosis in patients with nanophthalmos. Third, our focus solely on patients with nanophthalmos with secondary ACG restricts the comprehensiveness of the clinical spectrum we can cover. Fourth, as there was no CRB1 or BEST1 variant patient in our study, we failed to compare the clinical features of all the genotypes.

In conclusion, our study provided a detailed overview of the gene variant spectrum and clinical landscape of Chinese patients with NSACG, and explored genotype-phenotype correlations in depth. Patients with NSACG with a genetic diagnosis displayed shorter AL with a proportionally shorter ACD and a disproportionately shorter VCD, indicating that gene variants may affect vitreous expansion. AD and AR variants seemed to impact different stages of ocular development, leading to distinct phenotypes. Increased anterior segment crowding and potentially more easily expandable uvea in those with a genetic diagnosis may explain faster ACG progression. Whereas TMEM98 and MYRF variants might independently contribute to optic nerve damage, making early intervention is crucial to prevent severe outcomes in these patients. This study might open new pathways for exploring the molecular mechanisms underlying gene-related NSACG and for improving disease management and treatment strategies.