Unique Haplotypes in OPN1LW as a Common Cause of High Myopia With or Without Protanopia: A Potential Window Into Myopic Mechanism

Abstract

Purpose: Specific haplotypes (LVAVA, LIVVA, and LIAVA) formed by five polymorphisms (p.L153M, p.V171I, p.A174V, p.I178V, and p.S180A in exon 3 of OPN1LW) that cause partial or complete exon skipping have been reported as unique genetic causes of high myopia with or without colorblindness. This study aimed to identify the contribution of OPN1LW to early-onset high myopia (eoHM) and the molecular basis underlying eoHM with or without colorblindness.

Methods: Comparative analysis of exome sequencing data was conducted for 1226 families with eoHM and 9304 families with other eye conditions. OPN1LW variants detected by targeted or whole exome sequencing were confirmed by long-range amplification and Sanger sequencing, together with segregation analysis. The clinical data were thoroughly analyzed.

Results: Unique haplotypes and truncation variants in OPN1LW were detected exclusively in 68 of 1226 families with eoHM but in none of the 9304 families with other visual diseases (P = 1.63 × 10⁻⁶³). Four classes of variants were identified: haplotypes causing partial splicing defects in OPN1LW (LVAVA or LIVVA in 31 families), LVAVA in OPN1LW-OPN1MW hybrid gene (in 3 families), LIAVA in OPN1LW (in 29 families), and truncations in OPN1LW (in 5 families). The first class causes partial loss of red photopigments, whereas the latter three result in complete loss of red photopigments. This is different from the replacement of red with green owing to unequal re-arrangement causing red-green colorblindness alone. Of the 68 families, 42 affected male patients (31 families) with the first class of variants (LVAVA or LIVVA in OPN1LW) had eoHM alone, whereas 37 male patients with the latter 3 classes had eoHM with protanopia. Adaptive optics retinal imaging demonstrated reduced cone regularity and density in men with eoHM caused by OPN1LW variants compared to those patients with eoHM and without OPN1LW variants.

Conclusion: Based on the 68 families with unique variants in OPN1LW, our study provides firm evidence that the two different phenotypes (eoHM with or without colorblindness) are caused by two different classes of variants (partial splicing-effect haplotypes or complete splicing-effect haplotypes/truncation variants, respectively). The contribution of OPN1LW to eoHM (isolated and syndromic) was characterized by OPN1LW variants found in 5.5% (68/1226) of the eoHM families, making it the second most common cause of monogenic eoHM alone (2.4%) and a frequent cause of syndromic monogenic eoHM with colorblindness. Such haplotypes, in which each individual variant alone is considered a benign polymorphism, are potential candidates for other hereditary diseases with causes of missing genetic defects.

Color vision is determined by genes encoding three different cone photopigment types that were sensitive to long (OPN1LW), middle (OPN1MW), and short (OPN1SW) wavelengths.^1,2 OPN1LW and OPN1MW, located on Xq28 and arranged in a head-to-tail tandem array, share 98% sequence identity.^2,3 It is well known that unequal homologous recombination between OPN1LW and OPN1MW is frequently observed as the origin of the molecular defects that cause red-green color vision deficiency, the most common Mendelian trait in humans.^1–4 In addition, the inactivation of the OPN1LW and OPN1MW resulted in blue cone monochromacy (BCM).⁵ 

X-linked early-onset high myopia (eoHM) with colorblindness and/or cone dysfunction or other allied abnormalities, also called Bornholm eye disease, has been mapped to Xq28 and has been well-studied clinically and genetically.^6–8 Surprisingly, this syndrome is caused by unique haplotypes formed by a combination of 5 polymorphic missense variants (p.L153M, p.V171I, p.A174V, p.I178V, and p.S180A) in exon 3 of OPN1LW (LVAVA and LIAVA).^8–11 Subsequent studies demonstrated that LVAVA leads to the partial skipping of exon 3 (a mosaic truncation effect), whereas LIAVA leads to the complete skipping of exon 3 (similar to truncation variants).^12–14 Similarly, eoHM with colorblindness can be caused by truncation variants in OPN1LW.¹⁵ Recently, a novel haplotype LIVVA in OPN1LW that causes a partial splicing effect was also found to cause X-linked cone dystrophy with severe myopia.¹⁶ X-linked eoHM alone, without colorblindness or other allied anomalies, was also mapped to Xq28 and was shown to result from the LVAVA haplotype in OPN1LW in our previous studies.^15,17 One of our recent studies suggested that LVAVA haplotypes in OPN1LW may be the second most common cause of eoHM after ARR3.¹⁸ 

It is interesting to know the underlying molecular basis through which some haplotypes cause eoHM alone, whereas others cause eoHM with colorblindness (or Bornholm eye disease). Moreover, if there is a difference in terms of genetic defects, what is the proportion of eoHMs with and without colorblindness? In the current study, based on the analysis of targeted and whole exome sequencing data with variants detected in OPN1LW in our laboratory, the haplotypes of OPN1LW were thoroughly analyzed in probands from 1226 families with syndromic or nonsyndromic eoHM and compared with probands from 9304 families with other eye conditions. Unique variants of OPN1LW were confirmed using long-range amplification, Sanger sequencing, and segregation analysis. The clinical data were thoroughly evaluated, particularly refractive status, fundus changes, and color vision. Overall, 4 classes of unique variants were detected in 68 of the 1226 families with eoHM but in none of the 9304 families with other eye conditions. These included haplotypes that cause a partial splicing defect in OPN1LW (LVAVA or LIVVA, in 31 families), LVAVA in L-M hybrid gene (in 3 families), a complete splicing defect haplotype (LIAVA) in OPN1LW (in 29 families), and truncations in OPN1LW (in 5 families). Of these, 42 male patients from 31 families with partial splicing-effect haplotypes in OPN1LW had eoHM alone, and 37 male patients from the remaining 37 families had eoHM with protanopia. This study, based on a large cohort of Chinese patients with unique variants of OPN1LW, further confirmed that different OPN1LW haplotypes are associated with eoHM alone and eoHM with colorblindness. Furthermore, our data suggested that unique OPN1LW variants are a common cause of both eoHM alone and syndromic eoHM. This identification of unique haplotypes as a common cause of distinct phenotypes in a gene well-known for other diseases reminds us that cis combinations of individually benign polymorphisms should be explored as a potential cause for other hereditary diseases with “missing” genetic defects. 

Methods

Subjects

This study was approved by the Institutional Review Board of Zhongshan Ophthalmic Center. A total of 1226 families with eoHM and 9304 unrelated probands with other eye conditions were recruited from the Pediatric and Genetic Clinic of the Zhongshan Ophthalmic Center, Guangzhou, China. Written informed consent was obtained from the participants or their guardians in accordance with the tenets of the Declaration of Helsinki. Peripheral venous blood samples were obtained, and genomic DNA was isolated using previously described methods.¹⁹ The clinical data of each participant were collected, and all participants underwent a comprehensive ocular examination. 

Genetic Analysis

Targeted exome sequencing (TES) and whole exome sequencing (WES) were performed on leukocyte genomic DNA samples from all 1226 eoHM probands and 9304 unrelated probands with other ocular disorders, as previously described.^20–22 Based on the exome sequencing data, 5 significant polymorphic variants in exon 3 of OPN1LW (p.L153M, p.V171I, p.A174V, p.I178V, and p.S180A) were screened, and their combination formed high-risk haplotypes in OPN1LW. Individuals with one of the five high-risk OPN1LW haplotypes in hemizygous status that have been reported to cause vision disorders,^16,23 including probands with LIAVA haplotype (p.V171I, p.I178V, and p.S180A but not p.L153M and p.A174V), individuals with LVAVA (p.I178V and p.S180A but not p.L153M, p.V171I, and p.A174V), individuals with LIVVA haplotype (p.V171I, p.A174V, p.I178V, and p.S180A but not p.L153M), probands with the LIAVS haplotype (p.V171I and p.I178V but not p.L153M, p.A174V, and p.S180A), and individuals with MIAVA (p.L153M, p.V171I, p.I178V, and p.S180A but not p.A174V), were selected from the exome sequencing data. In addition, probands detected with truncation variants in OPN1LW by TES or WES were chosen. Probands detected with one of the five high-risk haplotypes mentioned above or truncation variants in OPN1LW in hemizygous status by TES or WES were further confirmed using a previously reported method that uses long-range polymerase chain reaction (PCR) to specifically amplify the first gene of the opsin gene arrays.^15,24 Long-range PCR was performed using the LA Taq Hot Start protocol (Takara, Kyoto, Japan). The product of the long-range PCR was used as the template in a second PCR reaction to amplify exons 2 to 5, as described previously.¹⁵ Finally, Sanger sequencing was used to determine the sequence of the first gene and any variants (Supplementary Fig. S1).²⁰ 

Clinical Assessment

The clinical data of the affected male patients with the LVAVA haplotype, LIVVA haplotype, LIAVA haplotype, and truncation variants were reviewed in detail, including the age of onset, age at examination, chief complaint, other symptoms, and family history. A comprehensive ocular evaluation was performed, including refractive errors, best-corrected visual acuity (BCVA), color vision function tests (Ishihara plates and Farnsworth-Munsell Dichotomous D-15 Test), axial length, color fundus photography, fundus autofluorescence (FAF), wild-field scanning laser ophthalmoscopy (SLO; Optos, UK, and ZEISS CLARUS 500, Germany), optical coherence tomography (OCT), and electroretinography (ERG), in accordance with the International Society for Clinical Electrophysiology of Vision standard. 

Adaptive Optics Retinal Imaging

The status of the cone photoreceptors located in the foveal and parafoveal areas was obtained and analyzed using an Adaptive Optics (AO) retinal camera (rtx1; Imagine Eyes, Orsay, France). At least 5 retinal imaging scans were acquired, including one foveal area and 4 perifoveal areas (temporally, nasally, superiorly, and inferiorly) of the retina, which was 2 degrees (approximately 540–600 µm) away from the foveal center with a standardized 80 × 80 µm sampling window size. The images captured from each participant were analyzed using the AO detect processing software, including cone density, regularity, dispersion, and spacing. 

Statistical Analysis

Statistical analysis was performed using SPSS Statistics 25.0, and P values less than 0.05 were considered statistically significant. The Mann-Whitney U test was performed to compare refractive errors between affected male patients with partial splicing effects (LVAVA and LIVVA) in OPN1LW and those with completely splicing effects (LIAVA) or truncation variants in OPN1LW. The χ² test was conducted to compare the impaired level of cones between the affected male patients with the haplotype having a partial skipping impact and those with variants causing complete loss of L-cone function. The unpaired t test was performed to compare the parameters of cone mosaic status analyzed by AO detect between affected male patients with unique variants in OPN1LW, female carriers with variants in OPN1LW in heterozygous status, and controls who were matched for age, sex, refraction, and BCVA but did not carry high-risk haplotypes in OPN1LW. The correlation between cone density and BCVA in affected male patients was determined using Spearman's correlation analysis. 

Results

Identification of Families With Unique Variants in OPN1LW

To gain a comprehensive understanding of the genetic and clinical features of OPN1LW-associated eoHM, three eoHM families in our previous study were included.¹⁵ Based on the analysis of the TES and WES data of both 1226 eoHM probands and 9304 probands with other ocular conditions, 68 probands were found with high-risk haplotypes or truncation variants in OPN1LW, including 26 detected with LVAVA (p.[L153; V171; A174; V178; A180]), 29 detected with LIAVA (p.[L153; I171; A174; V178; A180]), 8 detected with LIVVA (p.[L153; I171; V174; V178; A180]), and 5 probands with truncation variants (details below). Unique variants in OPN1LW were found in 68 probands of the 1226 families with eoHM but none of the 9304 families with other eye diseases (P = 1.63 × 10⁻⁶³). The 68 probands from the 1226 eoHM families were further confirmed by long-range PCR, secondary PCR, and Sanger sequencing of the entire OPN1LW gene, which identified 4 classes of unique haplotypes in a structurally intact opsin gene cluster (Fig. 1A). The LVAVA and LIVVA haplotypes in OPN1LW, which produce large amounts of aberrantly spliced transcripts while preserving low levels of correctly spliced red photopigment transcripts, were identified in 31 families (23 with LVAVA and 8 with LIVVA; Fig. 1B). The LVAVA in L-M hybrid gene (exons 2 and 3 belonging to OPN1LW and exons 4 and 5 belonging to OPN1MW), which encodes photopigments with a peak spectral sensitivity of green and produces a low level of functional photopigments because of the incomplete splicing impact of LVAVA, was identified in three families (Fig. 1C). The LIAVA haplotype of OPN1LW, in which exon 3 was aberrantly spliced and produced no functional red photopigments, was detected in 29 families (Fig. 1D). The truncation variants of OPN1LW were confirmed in five families (Fig. 1E). None of the five truncation variants, including one previously reported variant (c.617_620dup/p.F208Rfs*51) or four novel variants (c.427_433dup/p.A145Vfs*23, c.518_519insC/p.A174Cfs*84, c.739C>T/p.R247*, and c.757C>T/p.Q253*) were present in the gnomAD database. These unique variants in OPN1LW were verified by Sanger sequencing and confirmed by co-segregation in 45 families (15 with LVAVA in OPN1LW, 5 with LIVVA in OPN1LW, 20 with LIAVA in OPN1LW, 3 with LVAVA in L-M hybrid, and 2 with truncations; Fig. 2, Supplementary Figs. S2–S6). Classical X-linked recessive inheritance was observed in 47 families. No hemizygous individuals with MIAVA or LIAVS haplotypes were identified. 

Figure 1.

 

Structure of the four classes of unique variants in OPN1LW identified in our study. (A) Wild-type OPN1LW and OPN1MW are arranged in a head-to-tail tandem array on chromosome Xq28, encoding red and green cone photopigments maximally sensitive to long and middle wavelengths, respectively. Intact opsin arrays consisting of one OPN1LW with single nucleotide polymorphisms (p. [L153; V171; A174; I178; S180]) in exon 3 and one or more OPN1MW genes with only the first normally expressed are responsible for normal trichromatic vision. (B) LVAVA (p. [L153; V171; A174; V178; A180]) and LIVVA (p. [L153; I171; V174; V178; A180]) with a partial splicing defect in OPN1LW causing functional loss of most L-cone red photopigment and a few reserved transcripts encoding functional red opsin (mosaics status). (C) LVAVA in L-M hybrid gene (exons 2 and 3 belonging to OPN1LW while exons 4 and 5 belonging to OPN1MW) with amino acids in exon 5 of OPN1MW, which are important for most spectral sensitivity, leads to no functional red photopigment and partially damaged M cones due to the LVAVA haplotype. (D) LIAVA (p. [L153; I171; A174; V178; A180]) with complete splicing in OPN1LW causes no functional L-cones. (E) Truncation mutations in exon 3, exon 4, and exon 5 of OPN1LW results in the identical lack of red signaling as the LIAVA haplotype, causing the complete aberrant splicing and premature termination. The FG and E6 were primers for amplifying the first gene of the opsin array.

Figure 2.

 

Pedigrees of 65 newly identified families with unique variants in OPN1LW. Twenty-one families were identified with the LVAVA haplotype in OPN1LW, 8 families were identified with the LIVVA haplotype in OPN1LW, 29 families were identified with the LIAVA haplotype in OPN1LW, 3 families had the LVAVA haplotype in L-M hybrid gene, and 4 families had truncation variants in OPN1LW. The squares represent male individuals, and the circles represent female individuals. The filled squares and circles indicate affected male and affected female patients, respectively. Heterozygous female carriers are indicated by a circle with a dot in the center. A half-filled circle indicates a female carrier with high myopia in one eye. Probands in each family are indicated by arrows. The family numbers are located above the pedigrees.

Clinical Characterization

All 79 male patients carrying hemizygous variants in OPN1LW from the 68 families were diagnosed with eoHM, including 42 male patients from 31 families with partial splicing-effect haplotypes in OPN1LW who had eoHM alone and 37 male patients with the latter three types who had eoHM and protanopia. All 79 male patients complained of blurred vision since early childhood; however, none showed nystagmus, severe photophobia, or night blindness. Among the 77 affected male patients with refraction data, 89.6% (69/77) had a refractive error of ≤−5.00 diopters (D; or axial length ≥25 mm) before 7 years of age, whereas the remaining 6.5% (5/77) who were older than 18 years at their first visit to our clinic had refractive errors ≤−10.00 D. The available equivalent spherical data of all affected males ranged from −3.75 D to −22.00 D (median = –9.38 D, interquartile range [IQR] = approximately −7.13 D to −11.09 D; Supplementary Fig. S7A), in whom three male patients had moderate myopia in early visit but develop myopia to −5.00 D or greater in follow-up visit by age of 7 years. For example, follow-up refraction recordings were accessible for 20 affected male patients, showing a gradually increased trend with age up to 15 years of age (see Supplementary Fig. S7B). Two (8928-IV4 and 18972-II2) had equivalent spherical refractions of approximately −4.00 D at first presentation when they were less than 4 years old, but with progression reached −6.00 D by the age of 6 years. There were no differences in equivalent spherical diopters between male patients with partial splicing-effect haplotypes in OPN1LW (LVAVA and LIVVA) and those with loss of function variants in OPN1LW (LIAVA and truncations; see Supplementary Fig. S7D). The astigmatism diopter values were available from 75 of the 77 affected male patients, ranged from −0.25 D to −5.00 D (median = −2.00 D, IQR = approximately −1.25 D to −3.00 D), and 60 of them (80.0%) had astigmatism ≤−1.00 D. BCVA was available for 55 affected male patients and ranged from 0.04 (in one patient with retinal detachment) to 1.0 (median = 0.5, IQR = 0.4–0.7), with 85.5% (47/55) obtaining a BCVA no less than 0.3 (see Supplementary Fig. S6C). The remaining 8 male patients with BCVA less than 0.3 included 5 children younger than 4 years of age and 3 individuals over 35 years old. Color vision testing was conducted on 57 affected male patients, of whom 31 with a partial splicing-effect haplotype in OPN1LW had normal color vision (26 with LVAVA and 5 with LIVVA), and the remaining 25 affected male patients (18 with LIAVA in OPN1LW, 3 with LVAVA in a hybrid gene, and 4 with truncation variants) had protanopia. 

All 73 affected male patients with available posterior color fundus photographs presented with high myopia changes and were assessed according to the META-PM classification.²⁵ A relatively normal posterior retina without significant myopic changes (C0) was observed in 6.8% (5/73) of the male patients (Figs. 3A, 3B). Tessellated changes were concentrated in the posterior retina and more severely around the optic nerve, with visible choroidal vessels (C1) observed in 52.1% (38/73) of the male patients (Figs. 3C, 3D). Yellow-white diffuse chorioretinal atrophy, mainly in the area between the optic nerve and the macula (C2), was found in 34.2% (25/73) of the affected male patients (Figs. 3E, 3F). Well-defined white atrophic patches around the optic nerve (C3) were observed in 6.8% (5/73) of patients. Among the 20 male patients with FAF, 70.0% (14/20) had normal autofluorescence (Fig. 3G) and 30.0% (6/20) showed relatively hyper-autofluorescent changes in the macular region (Figs. 3H, 3I). Thirty-four male patients underwent wide-field fundus examinations, of whom 76.5% (26/34) presented with pigmentation changes at the boundary or a pigmentary disorder in the periphery (Figs. 3H, 3I), 5.9% (2/34) showed lattice-like degeneration, and the remaining 17.6% (6/34) had a normal-appearing peripheral retina. Different degrees of peripheral pigmentary degeneration may manifest differently when viewed using different imaging techniques. Some affected male patients, although showing peripheral pigmentary disturbances in SLO, appeared on color fundus images to have tessellated changes with no pigmentary changes and showed relatively normal autofluorescence on FAF (Figs. 3J-L, Supplementary Fig. S8). 

Figure 3.

 

Posterior color fundus photography, autofluorescence images, and wide-field fundus examinations of patients with unique variant types of OPN1LW as indicated in the plates. (A, B) A few patients showed a relatively normal-appearing fundus (C0). (C, D) Most patients presented with tessellated changes in the posterior retina and normal-appearing autofluorescence (C1). (E, F) Some patients had slightly yellowish-white chorioretinal atrophy, especially around the optic nerve (C2). Hyper-autofluorescent changes in the macular region were observed in some patients with C2 myopic fundus changes. (G) Two-thirds of the patients with a wide-field fundus examination showed tessellated fundus changes with corresponding normal autofluorescence. (H, I) The remaining patients presented with borderline pigmentary changes in the peripheral retina, observed as hyper-hypo autofluorescence changes on wild-field fundus autofluorescence images. (J-L A few patients showed obvious peripheral pigmentary degeneration on wild-field scanning laser ophthalmoscopy, which was not observed (J and K) or appeared relatively milder (L) on color fundus photography and showed normal autofluorescence. The right parts of the J to L images correspond to magnified images of the peripheral retinal area, in which the upper is captured by wild-field scanning laser ophthalmoscopy, the middle shows an enlarged color fundus, and the lower shows fundus autofluorescence.

Among the 41 affected males with OCT results, 70.7% (29/41) had normal macular foveal structures, six male patients (five with LIAVA and one with LIVVA) (14.6%) showed thinner outer nuclear layers and disrupted ellipsoid zones, and six male patients older than ten years (14.6%) presented with pathologic myopic macular degeneration (Supplementary Fig. S9). There were no statistically significant differences in the fundus changes or OCT structures between the males with eoHM alone due to the partial splicing-effect haplotypes in OPN1LW and those with the latter three types of variants leading to eoHM and protanopia. ERG examinations were available for 11 male patients with the LVAVA haplotype, including 9 with LVAVA in OPN1LW and 2 with LVAVA in the L-M hybrid gene, 5 of whom had mild cone involvement and normal rod responses, 5 had moderately reduced cone responses and normal or mildly reduced rod responses, and a 40-year-old man had severely reduced cone responses and moderate rod involvement. Two affected male patients with LIVVA presented with severe cone involvement and mildly reduced or normal rod responses. Eighteen male patients with LIAVA or truncation variants underwent ERG examinations, of which moderate cone impairment with normal rod responses was observed in 3 patients, and severely reduced cone responses with various degrees of reduced rod responses were found in 15 male patients (8 with normal or mildly reduced rod responses, 3 with moderate rod involvement, and 4 with severe rod impairment). Statistically significant differences were found in the level of cone impairment between male patients with partial splicing-effect haplotypes and those with loss of function variants (χ² test, P = 0.008). A total of 53 obligate female carriers were confirmed as heterozygous carriers, of whom 17 had high myopia at an early age (6 had high myopia in one eye only), with 32.1% (17/53) penetrance. 

Assessment of Cone Mosaic Status

The mean values of the cone features were calculated and compared for cases (10 affected male patients and 6 female carriers carrying different types of variants in OPN1LW) and controls (4 male patients and 1 female patient) matched for age, refraction, and BCVA. Statistically significant differences were identified between the affected male patients and controls in terms of cone regularity (87.1 ± 5.9 vs. 91.5 ± 5.0, P = 0.003), density (14886.8 ± 2678.2 vs. 24203.2 ± 3612.1, P < 0.0001), dispersion (18.1 ± 4.6 vs. 9.1 ± 0.9, P < 0.0001), and spacing (9.1 ± 0.9 vs. 7.2 ± 0.5, P < 0.0001). Statistically significant differences were also found between affected male patients and female carriers, who had regularity = 89.9 ± 5.0 (P = 0.03), density = 18644.2 ± 3612.1 (P < 0.0001), dispersion = 15.3 ± 4.8 (P = 0.01), and spacing = 8.2 ± 0.9 (P < 0.0001), as well as between female carriers and control individuals (P < 0.0001 for density and spacing; P = 0.002 for dispersion; Fig. 4). Additionally, no correlation was found between cone density and BCVA of the affected males (Spearman's correlation = 0.233, P = 0.578), reflecting the relatively narrow range of variation in which almost all had densities greater than those required for visual acuity greater than 0.3. 

Figure 4.

 

Adaptive optics (AO) retinal imaging of affected male patients with different types of unique variants in OPN1LW, heterozygous female carriers, and control individuals matched for sex, age, refraction, and BCVA to affected male patients or female carriers. All AO retinal imaging captured the cone cells at the 2 degrees temporal position of the central fovea. (A-D) Affected male patients with four classes of unique variants in OPN1LW presented with variable disruption of cone mosaic status. (E-H) Female carriers with different types of unique OPN1LW variants in heterozygous status presented with a relatively less disruptive cone mosaic state than affected male patients. (I-L) Control individuals matched for sex, age, refraction, and BCVA to affected male patients or female carriers with high myopia but without high-risk haplotypes or truncation variants in OPN1LW showed a relatively larger cone density. (M-P) Scatter diagrams comparing the regularity, density, dispersion, and spacing of cones between the affected probands, female carriers, and control individuals. The affected male patients showed decreased regularity and density but increased dispersion and spacing of cones compared with carriers and controls. The asterisks represent statistically significant differences: *, P < 0.05; **, P < 0.01; ***, P < 0.001, ****, P < 0.0001.

Discussion

In this study, unique variants in OPN1LW were detected exclusively in 68 of the 1226 families with eoHM but not in other ocular diseases, which was further confirmed by co-segregation analysis. High myopia is a constant phenotype in all affected male patients with unique haplotypes and truncation variants in OPN1LW, and the presence of colorblindness is determined by the amount of residual functional red photopigments in the L-cones. Unlike partial splicing-effect haplotypes in OPN1LW that result in mosaics status with different types of L-cones due to partial exon skipping effects, LIAVA haplotype and truncation variants in OPN1LW cause complete loss of functional red pigmentations in all L-cones though the 5′-prime regions are preserved, and the L-M hybrid gene encoding photopigments with the spectral sensitivity of green are impacted by the LVAVA haplotype. Based on the largest Chinese cohort of unique variants of OPN1LW to date, our study provides firm evidence that the two different phenotypes are caused by two different classes of variants: eoHM alone with partially truncated OPN1LW is due to partial splicing-effect haplotypes (LVAVA or LIVVA), whereas eoHM with colorblindness results from completely truncated OPN1LW due to the complete splicing-effect haplotype (LIAVA) or nonsense/frameshift variants. The partial splicing-effect haplotype (LVAVA) in the L-M hybrid gene lead to eoHM with colorblindness due to changes in spectral sensitivity. In combination with our previous analysis, OPN1LW was found not only to be the top two most frequently implicated genes for nonsyndromic monogenic eoHM (2.4%), second only to ARR3 (3.1%),¹⁸ but might be among the top five common causative genes for syndromic monogenic eoHM when patients with colorblindness were included. This is the first time that such unique haplotypes in a well-known gene have been implicated as one of the most common causes of nonsyndromic eoHM. The cone mosaic status observed by AO retinal examination in this study was consistent with that of previous studies.^10,26,27 

Rearrangement of OPN1LW and OPN1MW has long been well-known to cause red-green color vision deficiency, a common monogenetic disease. Initially, protanopia was reported to be attributed to a single L-M hybrid gene or replacement of OPN1LW by OPN1MW, which results in a single or both opsin genes with green spectrum sensitivity.^4,28 Subsequently, a single L-M hybrid gene with an inactivating missense mutation (p.C203R) or truncation variant was shown to result in BCM, a severe phenotype caused by the complete absence of L and M cone function.^29–31 High homology between OPN1LW and OPNMW contributes to numerous polymorphisms with allele frequencies greater than 1% in general population.¹ Combination of 5 polymorphisms in exon 3 forms various haplotypes with exon skipping impacts to different degrees. In particular, LVAVA and LIAVA have been reported to be associated with X-linked high myopia with accompanying colorblindness.⁸ The LVAVA haplotype was first identified as the cause of nonsyndromic eoHM in our previous studies.¹⁵ Variations in OPN1LW showed extensive phenotypic heterogeneity, ranging from severe BCM through isolated color vision deficiency to eoHM with and without colorblindness. Moreover, the important role of OPN1LW in high myopia has been underestimated in the past. This study showed a high frequency of OPN1LW variants in high myopia, and the mechanism of colorblindness accompanying OPN1LW-associated eoHM was clearly elucidated. Although the LVAVA haplotype in the L-M hybrid gene and LIAVA and truncation variants in OPN1LW presented with intact 5′-prime regions, no functional red photopigments were produced. This is different from color vision deficiency due to the rearrangement of structurally intact opsin gene arrays that occurs through the replacement of red photopigments by green ones without loss of functional photopigments. Whether OPN1LW-associated eoHM is accompanied by colorblindness depends on the levels and spectral characteristics of correctly spliced opsin mRNA in the opsin gene arrays caused by the diverse haplotypes of OPN1LW.^12–14 In addition to variants in OPN1LW causing eoHM, high-risk haplotypes in the highly homologous OPN1MW gene have also been reported to be responsible for high myopia through the same mechanism as OPN1LW variants. The focus of this study on OPN1LW-associated eoHM may have underestimated the contribution of variants in OPN1LW/OPN1MW to high myopia, which may increase with the inclusion of families with haplotypes in OPN1MW and other potential high-risk haplotypes in the opsin array. 

Although our study focused on the genotype profiles and clinical characteristics of OPN1LW in male patients, it was also found that 17 of the 53 female carriers of heterozygous OPN1LW variants had eoHM, whereas the remaining female carriers were asymptomatic. Because OPN1LW-associated eoHM is inherited as a classic X-linked recessive trait, the manifestation of eoHM in a small number of female carriers might be achieved by variations in random X-inactivation, which is also thought to be responsible for some female carriers of other X-linked recessive retinal diseases (e.g. retinal degeneration due to RPGR) that show variable phenotypic severity. Perhaps more directly related, this has also been reported in one heterozygous female with BCM.^32–34 Significant asymmetric phenotypes between eyes have also been found in heterozygous female carriers of RPGR variants,³⁵ which may share the same mechanism as six female carriers who presented with eoHM in only one eye. As described in the study by Neitz et al.,²³ based on AO retinal imaging examination, the mechanism by which some female carriers exhibit eoHM might be similar to the cone mosaic mechanism by which affected male patients with the OPN1LW variant develop eoHM, that is, mosaic cones with different amounts of photopigments mimicking defocus signals to bipolar cells and related neuronal networks influence the onset of high myopia. Further research on female carriers with unique high-risk haplotypes in OPN1LW is expected in the future. 

Signals from cones are relayed to bipolar cells, then to ganglion cells that finally transmit impulses to the central nervous system, building an intricate “microcircuit” in the retina. The wiring of this circuitry is private in the central fovea, an L or M cone privately directs input to an ON and OFF midget circuit, which allows high acuity.^36–38 Opposed signals from OPN1LW and OPN1MW antagonistically interact with each other and thus form the red-green opponent pathways underlying color vision.^39–41 A widely accepted receptive field in the central retina consists of segregated signals from different cones, including an excitatory central signal from an isolated L or M cone and inhibitory surrounding signals of nonselective input from different cones through horizontal cells.^36,42–45 High-spatial frequency luminance stimuli from the center opposed with the low-spatial frequency brightness stimuli from the neighbor wirings in the foveal private line midget circuit form the contrast signals which have been found to appear in the early fetal period⁴⁶ and to impact the growth process of the eyeball.^26,47–49 Individuals with intact opsin arrays have normal trichromatic vision and emmetropization processes (Fig. 5A, Supplementary Fig. S10A). When the original L-cones were replaced by cones with photopigments whose maximum spectral sensitivity was green, the spatial contrast in the visual process was unaffected, although protanopia occurred (Fig. 5B, Supplementary Fig. S10B). A partial splicing-effect haplotype in OPN1LW resulted in the various opsin properties in the adjacent cones of a single neighborhood, causing abnormal contrast and stimulating eyeball elongation (Fig. 5C, Supplementary Fig. S10C). When an aberrant mosaic status due to the LVAVA haplotype occurs in L-M hybrid gene,⁵⁰ both eoHM and protanopia occur (Fig. 5D, Supplementary Fig. S10D). LIAVA and truncations in OPN1LW resulted in the complete absence of functional cones with red photopigments, which severely damaged the spatial contrast and led to the loss of the ability to sense red light (Fig. 5E, Supplementary Figs. S10E, S10F). This contrast theory is thought to be the potential causal mechanism not only for OPN1LW-associated eoHM but also for common juvenile-onset myopia, which has been supported by clinical trials.^47,51 The same mechanisms are thought to underlie the development of high myopia in ARR3-associated MYP26 with X-linked female limited inheritance. Thus, the expression of both ARR3 and OPN1LW was cone-specific. Decreased levels of arrestin encoded by ARR3, functioning as the desensitizer of opsins, result in increased activity of L and M cones and enhanced sensitivity of color vision signals, which has been proposed to explain the onset of high myopia secondary to heterozygous variants in ARR3.⁵² It was hypothesized that the mosaic cone status with adjacent cones expressing different functional opsins of affected female patients with heterozygous variants in ARR3 is similar to that of affected male patients with hemizygous LVAVA in OPN1LW with a partial effect on splicing. The relationship between OPN1LW and ARR3 was similar to that between RHO and SAG, in which RHO, which encodes rhodopsin, was activated when retinal was isomerized from the 11-cis to the all-trans configuration, and its inactivation could be prevented by binding to the arrestin encoded by SAG.⁵³ 

Figure 5.

 

The mixed surround models for red-green spectral opponent explain the potential pathogenesis of eoHM resulting from the four classes of variants in OPN1LW. (A) In the central retinal area of humans, each cone has a “private line” circuit pathway; that is, each L or M cone relays signals to an ON midget bipolar cell and an OFF midget bipolar cell, then the bipolar cells further deliver the signal to midget ganglion cells, which form the excitatory center of the receptive field. The H1 horizontal cells non-selectively receive signals from the L and M cones in the central cone neighbor and form the inhibitory surrounded signal of each receptive field. Using the L-cone as the center, for example, signals conducted by this L-cone serve as the center signal, and the signals received by the surrounding L and M cones as opposed signals. The central and surrounded signals form the contrast that affects eye growth. (B) The midget circuit diagram for individuals with protanopia due to the presence of L-M hybrid gene. Although the L-cones are replaced with cones that are maximally sensitive to middle-wavelength light, the generation of excitatory and inhibitory signals are not disrupted. (C) In the presence of the partial splicing-effect haplotype in OPN1LW, most L-cones lose their functional pigmentation, and only a few normally expressed L-cones are retained. When the normally expressed L-cones act as the central signal in the receptive field, the surrounding inhibitory signal is affected by the absence of functional L-cones in the vicinity. The abnormal contrast signal between the central and surround of the receptive field produces blurred vision simulating a refractory error and stimulates the elongation of the eyeball. (D) The result of the LVAVA haplotype in hybrid L-M gene can be considered a combination of the consequences of a spectrally sensitive change due to the L-M hybrid gene and an abnormal spatial contrast signal caused by the LVAVA haplotype in L-M gene. (E) The LIAVA haplotype and truncation variants in OPN1LW lead to the complete loss of functional L-cone photopigment. When a nonfunctional L-cone acts as the central signal in the receptive field, the surrounding inhibitory signal is also affected by the absence of functional L-cones in the neighborhood, which results in early-onset high myopia with protanopia. LOF, loss of function.

In conclusion, variants in OPN1LW are a common cause of Mendelian eoHM with or without colorblindness, second only to ARR3 mutations in isolated eoHM. The proposed mechanism of OPN1LW-associated eoHM with or without colorblindness is that variants cause different amounts of functional photopigments, which result in mosaic and disproportional L-cones, causing abnormal spatial contrast signals to connect bipolar cells and more distal parts of the associated neuronal network. Proposing a similar mechanism for the top two most frequently implicated genes for eoHM, ARR3, and OPN1LW, emphasizes the potential significance of L-M cones in the myopic mechanism, which may also function in common juvenile-onset myopia. Further studies on cone-bipolar and surrounding neuronal connections in humans or nonhuman primates, may provide a valuable starting point for delineating myopic molecular mechanisms. Additionally, combinations of individually benign variants in the same gene, such as haplotypes in OPN1LW and beyond, may underlie a wide variety of diseases, warranting attention to inherited diseases that lack apparent genetic mechanisms. 

Acknowledgments

Funded by the National Natural Science Foundation of China (81770965, 81600768), the Science and Technology Planning Projects of Guangzhou (202102010271), and the Fundamental Research Funds of the State Key Laboratory of Ophthalmology. The sponsor or funding organization had no role in the design or conduct of this research. 

Contributors: X.X., S.L., X.J., W.S., and Q.Z. recruited the individuals diagnosed with different forms of ocular conditions and collected the clinical records. S.L., X.X., and X.J. prepared the genomic DNA from venous blood. X.X., S.L., P.W., and Q.Z. performed whole exome analysis and targeted exome sequencing. Y.W., Y.J., J.O., J.W., Z.Y., P.W., and Q.Z. participated in the bioinformatics analysis of exome sequencing data and in review and classification of clinical data. Y.W. performed Long-range PCR and Sanger sequence. Q.Z. designed the study. Y.W. and W.S. wrote the manuscript. J.F.F.H. and Q.Z. critically revised the manuscript. All authors reviewed and approved the manuscript. Y.W., W.S., and X.X. contributed equally to this work. 

Disclosure: Y. Wang, None; W. Sun, None; X. Xiao, None; Y. Jiang, None; J. Ouyang, None; J. Wang, None; Z. Yi, None; S. Li, None; X. Jia, None; P. Wang, None; J.F. Hejtmancik, None; Q. Zhang, None