Unique Variants in OPN1LW Cause Both Syndromic and Nonsyndromic X-Linked High Myopia Mapped to MYP1

Abstract

Purpose.: MYP1 is a locus for X-linked syndromic and nonsyndromic high myopia. Recently, unique haplotypes in OPN1LW were found to be responsible for X-linked syndromic high myopia mapped to MYP1. The current study is to test if such variants in OPN1LW are also responsible for X-linked nonsyndromic high myopia mapped to MYP1.

Methods.: The proband of the family previously mapped to MYP1 was initially analyzed using whole-exome sequencing and whole-genome sequencing. Additional probands with early-onset high myopia were analyzed using whole-exome sequencing. Variants in OPN1LW were selected and confirmed by Sanger sequencing. Long-range and second PCR were used to determine the haplotype and the first gene of the red-green gene array. Candidate variants were further validated in family members and controls.

Results.: The unique LVAVA haplotype in OPN1LW was detected in the family with X-linked nonsyndromic high myopia mapped to MYP1. In addition, this haplotype and a novel frameshift mutation (c.617_620dup, p.Phe208Argfs*51) in OPN1LW were detected in two other families with X-linked high myopia. The unique haplotype cosegregated with high myopia in the two families, with a maximum LOD score of 3.34 and 2.31 at θ = 0. OPN1LW with the variants in these families was the first gene in the red-green gene array and was not present in 247 male controls. Reevaluation of the clinical data in both families with the unique haplotype suggested nonsyndromic high myopia.

Conclusions.: Our study confirms the findings that unique variants in OPN1LW are responsible for both syndromic and nonsyndromic X-linked high myopia mapped to MYP1.

Located in Xq28, MYP1 (OMIM 310460) is a locus for syndromic and nonsyndromic high myopia. It was first described in a large Danish family with syndromic high myopia (i.e., Bornholm Eye Disease), which was characterized by high myopia, cone dysfunction, and deuteranopic color vision defect.^1,2 

A Minnesota family of Danish origin with similar phenotype also was mapped to MYP1.³ An additional five families with related phenotypes were subsequently identified.^4,5 The genetic defects in MYP1 responsible for X-linked syndromic high myopia have not been clear. Until recently, unique haplotypes (LIAVA and LVAVA) in OPN1LW have been reported as being responsible for X-linked syndromic high myopia mapped to MYP1.⁵ In our previous study, a large family with X-linked nonsyndromic high myopia was mapped to MYP1.⁶ Thus, it would be of interest to know whether variations in OPN1LW also were responsible for X-linked nonsyndromic high myopia mapped to MYP1. 

The current study describes the identification of a unique haplotype (LVAVA) in OPN1LW in a large family with nonsyndromic high myopia previously mapped to MYP1,⁶ as well as in another large family with nonsyndromic high myopia. Our results confirm the involvement of a unique haplotype in OPN1LW as a cause of X-linked high myopia, both syndromic and nonsyndromic. 

Materials and Methods

Subjects

This study is a part of our ongoing project to study genetic defects involved in early-onset high myopia. Patients were recruited from the clinic of the Zhongshan Ophthalmic Center. Written informed consent was obtained from the participants or their guardians, following the tenets of the Declaration of Helsinki. This study was approved by the institutional review board of the Zhongshan Ophthalmic Center. Ophthalmologic examinations were performed by ophthalmologists of the Zhongshan Ophthalmic Center. Our inclusion criteria for early-onset high myopia were the same as previously described⁷: spherical refraction in each meridian equal to or greater than −6.00 diopters (D) in both eyes, high myopia developed before the age of 7 years, and no other known ocular or related systemic diseases. Color vision was tested with the Ishihara Test Plates. Revaluation of color vision was further performed on three available patients (HM295IV4, HM346III6, and HM429III3) using the Oculus Heidelberg anomaloscope (OCULUS, Lynnwood, WA, USA). Additional examinations including electroretinography, fundus fluorescein angiography, and optical coherence tomography were conducted on the available patients. 

Sequencing Analysis

Whole-exome sequencing (WES) and whole-genome sequencing (WGS) had been performed on the sample of the proband from the family mapped to MYP1.⁶ Additional probands with early-onset high myopia were analyzed using WES. Whole-genome sequencing was carried out by Macrogen (Shenzhen, China; http://www.macrogen.com/) and WES was carried out by BGI (Shenzhen, China; http://www.genomics.cn/en/index). Whole-genome sequencing was performed with a TruSeq DNA Sample Prep Kit (Illumina, San Diego, CA, USA) on an Illumina HiSequation 2000 with a sequencing depth of 30×. Whole-exon sequencing was performed with a NimbleGen SeqCap EZ Exome array (44Mbp, Roche NimbleGen, Madison, WI, USA) on an Illumina Genome Analyzer II platform with sequencing depth of 50× or 100×. The quality controls of WGS and WES were previously described.^8,9 Variants in OPN1LW were selected from the data obtained from WGS and WES, and confirmed by Sanger sequencing. The methods used for amplification, sequencing, and analysis of the target fragments were previously reported.¹⁰ The descriptions of the variants are consistent with the nomenclature for sequence variations (Human Genome Variation Society, http://www.hgvs.org/mutnomen/).¹¹ The primers used to amplify these fragments are listed in Supplementary Table S1 and were designed using the Primer3 online tool (http://bioinfo.ut.ee/primer3-0.4.0/). 

Long-range and Second PCR

As previously described,¹² the pair of primers (first gene) were specific to the first gene in the red-green gene array (Supplementary Table S1). Primer-specific long-range PCR was used to amplify the first gene in the red-green gene array. The reaction was conducted according to the protocol for the Tks Gflex DNA Polymerase (Takara, Kyoto, Japan). The amplified product was purified and then used as a template in the second PCR to amplify exon 2 to exon 5 using exon-specific primers (Supplementary Table S1). All PCR products were sequenced as in our previous study¹⁰ to determine the haplotypes and the first gene of the red-green gene array. 

Results

For the proband from family HM295 with nonsyndromic high myopia mapped to MYP1, no novel candidate variant was detected based on comprehensive analysis of the data from WGS and WES. However, two rare variants (c.532A > G and c.538T > G) in OPN1LW, the key elements for the unique haplotype (LVAVA) were detected in the proband. Primer-specific long-range and second PCR confirmed that these two rare variants were present in the first OPN1LW gene and formed the unique haplotype (LVAVA) with other adjacent polymorphisms (Fig. 1A), which encoded a combination of amino acids across sites 153, 171, 174, 178, and 180 (the common sequences at these amino acid sites are leucine, valine, alanine, isoleucine, and serine [LVAIS]). This unique haplotype cosegregated with X-linked high myopia in the family (Fig. 1B; Supplementary Table S2), with a maximum LOD score of 3.34 at theta = 0. 

Figure 1

 

Sequence chromatography and pedigrees of HM295 and HM346. (A) Sequence chromatography. Sequence changes of the rare haplotype (LVAVA) and the wild-type haplotype (LVAIS) align with wild-type sequences. Amino acid encoded by the nucleotides is shown at the bottom of the sequence chromatography. Numbers at the top of the Figure (171, 174, 178, and 180) represent the nucleotide sequences that encode amino acids across sites 171, 174, 178, and 180 of either Val, Ala, Val, Ala, or Val, Ala, Ile, Ser. (B, C) Pedigrees of HM295 and HM346. WT or +, wild-type; M, LVAVA; arrow, proband; square, male; circle, female; blackened symbol, affected.

Analysis of data from WES on other probands with early-onset high myopia detected the two variants (c.532A > G and c.538T > G) in OPN1LW in one proband from a large family (HM346) and a frameshift mutation (c.617_620dup, p.Phe208Argfs*51) in OPN1LW in one proband from a small family (HM429). The long-range and second PCR also confirmed that the two variants in HM346 were present in the first OPN1LW gene and formed the unique haplotype (LVAVA) with other adjacent polymorphisms (Fig. 1A). The unique haplotype cosegregated with high myopia in the large family with a maximum logarithm of the odds (LOD) score of 2.31 at theta = 0 (Fig. 1C; Supplementary Table S2). Sanger sequencing confirmed the frameshift mutation in HM429 (Fig. 2). Segregation analysis was not available from the proband with the frameshift mutation. High myopia in these two additional families was transmitted as an X-linked trait. The unique haplotype (LVAVA) and the frameshift mutation (p.Phe208Argfs*51) in OPN1LW were not detected in 247 male controls. 

Figure 2

 

Sequence chromatography and pedigree of HM429. (A) Sequence chromatography. Forward and reverse sequence changes detected in the patient are shown on the top, whereas healthy control is shown at the bottom. The variation is marked with arrows and named under the sequence. (B) Pedigree of HM429. The variation is named under the pedigree. Arrow, proband; square, male; circle, female; blackened symbol, affected.

For the two families (HM295 and HM346) with the LVAVA haplotype, clinical data for subjects with high myopia in HM295 have been described previously,⁶ which indicated an early-onset nonsyndromic high myopia (Figs. 3A, 3G). Clinical data of available family members for HM346 are summarized in the Table. Ophthalmoscopic observation showed myopic fundus changes (Fig. 3B). Electroretinogram recordings on three patients (HM346II2, HM346III6, HM346IV2) showed normal rod responses and mild to moderate reduced cone responses, as shown in Figure 3H for the left eye of the patient HM346IV2. Four patients with high myopia in HM346 (HM346II4, HM346III1, HM346III6, and HM346IV2) were tested with the Ishihara Test Plates and found to have normal color vision (Table). Anomaloscope examination further identified HM346III6 with normal color vision (Fig. 3K) and HM295IV4 with a very mild protanomaly, a subtle form that was undetectable by the Ishihara Test Plates (Fig. 3J). Unlike the clinical features of X-linked syndromic high myopia, these findings suggested a nonsyndromic form of high myopia in the two families. 

Figure 3

 

Clinical features of patients with high myopia. Fundus photographs for left eyes of three patients (HM295IV1, HM346IV2, and HM429III3) demonstrate myopic fundus changes, including temporal crescent of optic nerve head and tigroid appearance of posterior retina (A–C). Fundus fluorescein angiography and optical coherence tomography for left eye of the patient HM429III3 show a retina with normal foveal contour (D–F). (D) and (E) show early and late phases of fundus fluorescein angiography, respectively. Electroretinography recordings demonstrate normal rod responses and mild to moderate reduced cone responses for patients HM295IV1, HM346IV2, and HM429III3 (G–I). Anomaloscopy for left eyes of patients show a very mild protanomaly in HM295IV4 (J), normal color vision in HM346III6 (K), and protanopia in HM429III3 (L). For (J–L), the yellow line and the blue dot represent the matching range for patients; the default background divided into six matching ranges represents different color vision conditions, including normal, protanopia, protanomaly, deuteranomaly, deuteranopia, and achromatopsia.

Table

 

Summary of Clinical Data in HM346 and HM429 Families

 

For the family (HM429) with the frameshift mutation, the proband was the only participant in this study. The clinical data of this proband are shown in the Table. Ophthalmoscopy observed myopic fundus changes (Fig. 3C). Fundus fluorescein angiography and optical coherence tomography demonstrated a retina with normal foveal contour (Figs. 3D–F). Electroretinogram recordings depicted normal rod responses and moderate reduced cone responses (Fig. 3I). Color vision tests (Ishihara Test Plates and anomoloscope) revealed protanopic color vision defect (Fig. 3L). 

Discussion

This study identified a unique haplotype (LVAVA) and a frameshift mutation (c.617_620dup) in OPN1LW in three families with X-linked high myopia; one of these families was previously mapped to MYP1.⁶ The OPN1LW with the variants was located in the first gene of the red-green gene array and was absent in 247 male controls. 

The MYP1 is the first locus for high myopia mapped to Xq28.^1,2 Affected individuals of the first family mapped to this locus had high myopia, amblyopia, and deuteranopia, with associated signs of optic nerve hypoplasia, reduced electroretinographic flicker, and nonspecific retinal pigment abnormalities, indicative of syndromic form of high myopia.^1,2 Subsequently, the genetic locus for individuals with similar phenotypes in additional families was identified^3–5 and mapped to MYP1.³ Sequencing analysis of a number of genes^4,13 in the linkage region did not identify the exact genetic defects until recently.⁵ 

Located in the MYP1 locus (Xq28), OPN1LW encodes one of the three cone pigments necessary for color vision. Unequal recombinations between OPN1LW and OPN1MW are the common cause of red-green color vision deficiency.^14–18 Deletion of the locus control region of the red-green gene array and a few unique point mutations are the main cause of blue cone monochromacy.^19,20 High myopia is not a frequent sign of color vision deficiency and blue cone monochromacy. This gene has not been considered as a candidate gene for high myopia thus far because of the close relationship of its variations with color vision deficiency and blue cone monochromacy. A recent study identified two unique haplotypes (LIAVA and LVAVA) in OPN1LW in six families with syndromic high myopia, including two families previously mapped to MYP1.⁵ In the present study, one unique haplotype (LVAVA) in OPN1LW cosegregates with X-linked nonsyndromic high myopia in two large families with a maximum LOD score of 3.34 and 2.31, respectively. One of these two families was mapped to MYP1 in our previous study.⁶ Overall, unique haplotypes in OPN1LW have been found exclusively in both syndromic and nonsyndromic X-linked high myopia mapped to MYP1. The pathogenicity of the LVAVA haplotype is unclear at present, but the similar haplotype LIAVA was reported to be deleterious due to a skipping of exon 3.^21,22 Further study is needed to determine whether LVAVA also has a potential effect on splicing, thereby producing a nonfunctional pigment and resulting in high myopia. 

The frameshift mutation (c.617_620dup, p.Phe208Argfs*51) in OPN1LW detected in the proband of HM429 has not been reported before and is absent in 1000 Genomes (http://browser.1000genomes.org/index.html) and Exome Variants Server (http://evs.gs.washington.edu/EVS/) databases. Screening of 247 male healthy controls does not detect the same variant. 

The c.617_620dup mutation is located in the early position of exon 4 in OPN1LW, which might produce a transcript ascribed to escaping from nonsense-mediated decay.^23–25 The proband was identified with high myopia and protanopia, but the genotype and phenotype in other affected relatives were unable to obtain. Therefore, the association between the genetic defect in OPN1LW and the phenotype in this family is hard to determine. 

The Ishihara Test Plates are widely used in clinical practice as the screening test to identify color vision deficiency. Severe color vision defect, either protanopia or deuteranopia, is the constant feature in X-linked syndromic high myopia and has been detected using the Ishihara Test Plates.³ Patients with high myopia in the current study had passed the screening test, so no sensitive color vision examinations had been performed at their first visit. However, the Ishihara Test Plates are a relatively insensitive cutoff test and are not sufficiently sensitive to detect subtle color vision defects. Whether there is an undetected subtle color vision defect is unclear. Therefore, the clinical data of families with the LVAVA haplotype only suggest a form of nonsyndromic high myopia. 

In summary, our results reveal the unique haplotype (LVAVA) in families with X-linked nonsyndromic high myopia and support the involvement of a unique haplotype in OPN1LW in the development of high myopia. These findings, taken together, suggest that the unique haplotype in OPN1LW is likely to be the common cause of both syndromic and nonsyndromic X-linked high myopia mapped to MYP1. 

Acknowledgments

The authors thank all of the patients and controls for their participation in this study. 

Supported by the National Natural Science Foundation of China (U1201221), Natural Science Foundation of Guangdong (S2013030012978), and the Fundamental Research Funds of the State Key Laboratory of Ophthalmology. 

Disclosure: J. Li, None; B. Gao, None; L. Guan, None; X. Xiao, None; J. Zhang, None; S. Li, None; H. Jiang, None; X. Jia, None; J. Yang, None; X. Guo, None; Y. Yin, None; J. Wang, None; Q. Zhang, None