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Assessing Foveal Structure in Individuals with TYR R402Q and S192Y Hypomorphic Alleles

Open AccessPublished:November 16, 2021DOI:https://doi.org/10.1016/j.xops.2021.100077

      Purpose

      To assess the impact of two TYR hypomorphic alleles (R402Q and S192Y) on foveal pit and foveal avascular zone (FAZ) morphology.

      Design

      Prospective, cross-sectional study.

      Participants

      A total of 164 participants with normal vision (67 male and 97 female; mean ± standard deviation [SD] age = 30.5 ± 12.8 years) were recruited.

      Methods

      Sequencing of more than 100 pigmentation-related genes was performed, and results were reviewed for the presence or absence of the TYR polymorphisms R402Q (rs1126809) and S192Y (rs1042602). Volumetric scans of the macula were obtained for each participant using OCT, and retinal thickness maps were analyzed using custom software. OCT angiography was used to image the FAZ, which was manually segmented and measured. Linear mixed model analysis was used to assess associations between genotype and foveal pit morphology.

      Main Outcome Measures

      Foveal pit depth, diameter, volume, and FAZ area in relation to the presence of hypomorphic alleles R402Q and S192Y on the TYR gene.

      Results

      Heterozygosity for the TYR R402Q allele was associated with decreased pit diameter (P = 0.0094) and decreased FAZ area (P = 0.025). Homozygosity for the TYR R402Q allele was associated with reduced pit volume (P = 0.0005), decreased pit depth (P = 0.007), reduced pit diameter (P = 0.0052), and reduced FAZ area (P = 0.0012). Homozygosity for TYR S192Y was associated with reduced FAZ area (P = 0.016). Heterozygosity for the TYR S192Y allele was not associated with differences in foveal pit depth, diameter, volume, or FAZ area (P > 0.05).

      Conclusions

      Although the role of the TYR R402Q and S192Y hypomorphic alleles in albinism remains controversial, our data suggest that these variants contribute to the extensive inter-individual variability in foveal morphology in the normal population. Our results contribute to the evolving picture of the relationship between ocular pigmentation and foveal morphology.

      Keywords

      Abbreviations and Acronyms:

      FAZ (foveal avascular zone), SD (standard deviation)
      The fovea is a highly specialized region of the primate retina and is responsible for high acuity vision. The foveal region is characterized by a foveal avascular zone (FAZ), increased cone photoreceptor density, and the characteristic pit (“fovea” is Latin for “pit”). Although the steps involved in development of the fovea have been well elucidated,
      • Bringmann A.
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      The primate fovea: structure, function and development.
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      one aspect of foveal anatomy that is not well understood is the enormous variation in the size of the pit and FAZ. For example, Wilk et al
      • Wilk M.A.
      • McAllister J.T.
      • Cooper R.F.
      • et al.
      Relationship between foveal cone specialization and pit morphology in albinism.
      showed that pit volume varied by 11-fold among 64 individuals with normal vision.
      • Wilk M.A.
      • McAllister J.T.
      • Cooper R.F.
      • et al.
      Relationship between foveal cone specialization and pit morphology in albinism.
      Chui et al
      • Chui T.Y.P.
      • Zhong Z.
      • Song H.
      • Burns S.A.
      Foveal avascular zone and its relationship to foveal pit shape.
      also found significant inter-individual variability, with a 2.5-fold variation in pit depth among 11 individuals with normal vision. Additionally, in a study of 110 normal foveae, Tick et al
      • Tick S.
      • Rossant F.
      • Ghorbel I.
      • et al.
      Foveal shape and structure in a normal population.
      reported large coefficient of variations for both pit depth (16.7%) and foveal inner retinal area (22.1%). Finally, Linderman et al
      • Linderman R.E.
      • Muthiah M.N.
      • Omoba S.B.
      • et al.
      Variability of foveal avascular zone metrics derived from optical coherence tomography angiography images.
      found FAZ area varied by 9-fold in a study with 350 normal foveae. Although conditions such as premature birth have been associated with smaller-sized pits and FAZs,
      • Ecsedy M.
      • Szamosi A.
      • Karkó C.
      • et al.
      A comparison of macular structure imaged by optical coherence tomography in preterm and full-term children.
      • Quinn G.E.
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      Outcome of prematurity and retinopathy of prematurity.
      • Akula J.D.
      • Arellano I.A.
      • Swanson E.A.
      • et al.
      The fovea in retinopathy of prematurity.
      the factors underlying normal variation in pit size remain incompletely understood.
      Insight on this issue comes from data on individuals with albinism. These individuals have absent or reduced retinal melanin and lack a fully developed foveal pit (i.e., foveal hypoplasia).
      • McAllister J.T.
      • Dubis A.M.
      • Tait D.M.
      • et al.
      Arrested development: high-resolution imaging of foveal morphology in albinism.
      ,
      • Thomas M.G.
      • Kumar A.
      • Mohammad S.
      • et al.
      Structural grading of foveal hypoplasia using spectral-domain optical coherence tomography: a predictor of visual acuity?.
      A further link between pigmentation and foveal anatomy has been suggested by literature comparing normal foveal anatomy between different racial groups. It has been shown that Black individuals have significantly deeper and broader pits compared with their White counterparts.
      • Wagner-Schuman M.
      • Dubis A.M.
      • Nordgren R.N.
      • et al.
      Race- and sex-related differences in retinal thickness and foveal pit morphology.
      ,
      • Zouache M.A.
      • Silvestri G.
      • Amoaku W.M.
      • et al.
      Comparison of the morphology of the foveal pit between African and Caucasian populations.
      Although there is no evidence for racial differences in retinal pigment epithelium melanin, there are racial differences in choroidal melanin.
      • Weiter J.J.
      • Delori F.C.
      • Wing G.L.
      • Fitch K.A.
      Retinal pigment epithelial lipofuscin and melanin and choroidal melanin in human eyes.
      If racial differences in choroidal melanin result in differences in foveal morphology, it stands to reason that individual variation in choroidal melanin could contribute to the described variation in foveal morphology.
      One of the key enzymes in the melanin biosynthesis pathway is tyrosinase, encoded by the TYR gene. Although TYR polymorphisms are known to cause autosomal recessive oculocutaneous albinism, there are some common hypomorphic alleles whose involvement in albinism is controversial.
      • Grønskov K.
      • Jespersgaard C.
      • Bruun G.H.
      • et al.
      A pathogenic haplotype, common in Europeans, causes autosomal recessive albinism and uncovers missing heritability in OCA1.
      • Norman C.S.
      • O'Gorman L.
      • Gibson J.
      • et al.
      Identification of a functionally significant tri-allelic genotype in the Tyrosinase gene (TYR) causing hypomorphic oculocutaneous albinism (OCA1B).
      • Oetting W.S.
      • Pietsch J.
      • Brott M.J.
      • et al.
      The R402Q tyrosinase variant does not cause autosomal recessive ocular albinism.
      • Monfermé S.
      • Lasseaux E.
      • Duncombe-Poulet C.
      • et al.
      Mild form of oculocutaneous albinism type 1: Phenotypic analysis of compound heterozygous patients with the R402Q variant of the TYR gene.
      Despite the controversy surrounding their pathogenicity, it has been shown that these alleles are associated with reduced enzymatic activity of tyrosinase.
      • Dolinska M.B.
      • Kus N.J.
      • Farney S.K.
      • et al.
      Oculocutaneous albinism type 1: Link between mutations, tyrosinase conformational stability, and enzymatic activity.
      For example, the R402Q variant (rs1126809) has been reported to have 25% to 67% enzymatic activity relative to wild-type at normal body temperature,
      • Dolinska M.B.
      • Kus N.J.
      • Farney S.K.
      • et al.
      Oculocutaneous albinism type 1: Link between mutations, tyrosinase conformational stability, and enzymatic activity.
      ,
      • Tripathi R.K.
      • Giebel L.B.
      • Strunk K.M.
      • Spritz R.A.
      A polymorphism of the human tyrosinase gene is associated with temperature-sensitive enzymatic activity.
      whereas the S192Y variant (rs1042602) results in 60% enzymatic activity.
      • Chaki M.
      • Sengupta M.
      • Mondal M.
      • et al.
      Molecular and functional studies of tyrosinase variants among Indian oculocutaneous albinism type 1 patients.
      The allele frequency of R402Q is 25% worldwide, but higher in European (non-Finnish) populations (36.4%).
      • Karczewski K.J.
      • Francioli L.C.
      • Tiao G.
      • et al.
      The mutational constraint spectrum quantified from variation in 141,456 humans.
      Likewise, the allele frequency of S192Y is 17.65% worldwide and 27.28% in European (non-Finnish) populations.
      • Karczewski K.J.
      • Francioli L.C.
      • Tiao G.
      • et al.
      The mutational constraint spectrum quantified from variation in 141,456 humans.
      The high frequency of these alleles, together with their mechanism of action, make them intriguing candidates for contributors to the normal variability in the size of the human fovea. We sought to examine the relationship between TYR genotype and the size of the pit and FAZ in individuals with normal vision.

      Methods

      Participants

      This study was approved by the Institutional Review Boards at the Medical College of Wisconsin (PRO23898) and followed the tenets of the Declaration of Helsinki. All participants provided informed consent following discussion of the risks of participation. A legal guardian provided consent for participants younger than 18 years of age. All participants completed an ocular health questionnaire and underwent color vision testing using the Neitz test of Color Vision (Western Psychological Services).
      • Neitz M.
      • Neitz J.
      A new mass screening test for color-vision deficiencies in children.
      By using the National Institutes of Health’s racial and ethnic categories, each participant was given the option to report their race and ethnicity. Exclusion criteria for the study included being aged less than 5 years, color vision deficits, and self-reported ocular, systemic disease, or being born prematurely.

      Genetic Testing

      Samples were sent to the Marshfield Clinic Research Foundation. Genomic DNA was extracted from the submitted specimen followed by enzymatic fragmentation and enrichment using SureSelect Custom DNA Target Enrichment Probes QXT (Agilent Technologies) that targeted more than 100 pigmentation-related genes. Size selection and cleanups were performed using KAPA Pure Beads (Roche); size selection and libraries were visualized on an Agilent 2100 Bioanalyzer. Libraries were quantified using KAPA Library Quantification Kit (Illumina) and visualized on a LightCycler 480 (Roche). Samples were 10-plexed/cartridge and pair-end sequenced (75 base pairs) on Miseq (Illumina). The sequenced data were aligned (GRCh37), variant-called, and annotated using SureCall v4.0, v4.1, v4.2 (Agilent Technologies).

      Foveal Morphology Imaging and Analysis

      Axial length was measured using the IOL Master 500 (Carl Zeiss Meditec). OCT imaging was performed on the right eye for each participant, using the Cirrus 6000 high-definition OCT (Carl Zeiss Meditec). Each volume scan was a nominal 6 × 6-mm scan consisting of 128 B-scans (512 A-scans/B-scan).
      Foveal pit morphology was analyzed using a previously published difference of Gaussian algorithm.
      • Wilk M.A.
      • McAllister J.T.
      • Cooper R.F.
      • et al.
      Relationship between foveal cone specialization and pit morphology in albinism.
      The retinal pigment epithelium internal limiting membrane thickness data file for each volume was imported into the software and was bilinearly interpolated to 512 × 512 pixels. The volume was then segmented into 1° radial slices passing through the foveal center (determined using the FoveaFinder algorithm on the Cirrus software). The thickness profile of each slice was fit to a difference of Gaussian model fit where all parameters varied freely to account for potential asymmetry of the retinal height and slope on either side of the fovea. Foveal metrics were measured on the basis of the Gaussian model fit values for each radial slice. Foveal pit depth was defined as the difference of the average maximum height and at the retinal thickness minimum. Foveal pit diameter was defined as the distance between the 2 maximum heights. Foveal pit depth and diameter were then averaged across all radial slices.
      To calculate the foveal volume, the different fits were all plotted together, and a fifth order polynomial was calculated to create a 1-dimensional contour filter that was then applied at the fovea (defined as the area between the maximum height). Once the smoothing was complete, the surface, or the top of the fovea, was calculated by drawing a line between the peaks of the 2 Gaussian curve fits. Once this was done, the surface was then overlaid onto the thickness measurements and the macular thickness measurements were subtracted from the surface to estimate the volume filling that space.

      Foveal Avascular Zone Imaging and Analysis

      With the Optovue’s Avanti system, OCT angiography was done on all but 6 participants. For all but 2 subjects who were imaged, 2 to 5 angiograms centered on the fovea were taken at a nominal scan size of 3 × 3 mm. For the 2 subjects, a single angiogram was used. For each angiogram, 2 volumes consisting of 304 B-scans at 304 A-scans/B-scan were acquired; 1 volume had a horizontal fast scanning axis, and the other volume had a vertical fast scanning axis. The 2 volumes were registered (AngioVue software version: 2018.1.0.43) to create a single angiogram volume with reduced motion artifacts and better signal-to-noise ratio. For each volume, the full-thickness slab (inner limiting membrane to 9 μm above the outer plexiform layer) was extracted. The 2 to 5 extracted images were then averaged together using bUnwarpJ
      • Arganda-Carreras I.
      • Sorzano C.O.S.
      • Marabini R.
      • et al.
      Consistent and elastic registration of histological sections using vector-spline regularization.
      in FIJI
      • Schindelin J.
      • Arganda-Carreras I.
      • Frise E.
      • et al.
      Fiji: An open-source platform for biological-image analysis.
      to further increase the signal-to-noise ratio and cropped to 304 × 304 pixels. The averaged image was used for further FAZ analysis.
      Using ImageJ’s multipoint tool,
      • Schneider C.A.
      • Rasband W.S.
      • Eliceiri K.W.
      NIH Image to ImageJ: 25 years of image analysis.
      a single observer (R.E.L.) segmented each FAZ. The segmentation coordinates were entered into a custom MATLAB script that has been previously described.
      • Linderman R.E.
      • Georgiou M.
      • Woertz E.N.
      • et al.
      Preservation of the foveal avascular zone in achromatopsia despite the absence of a fully formed pit.
      ,
      • Kraker J.A.
      • Omoba B.S.
      • Cava J.A.
      • et al.
      Assessing the influence of OCT-A device and scan size on retinal vascular metrics.
      The area of the FAZ (or the largest avascular area if the FAZ was fragmented
      • Linderman R.E.
      • Cava J.A.
      • Salmon A.E.
      • et al.
      Visual acuity and foveal structure in eyes with fragmented foveal avascular zones.
      ) was computed in pixels using the polyarea function in MATLAB. This was converted to mm2 by multiplying the nominal image scale (9.87 μm/pixel) by the ratio of the participant’s measured axial length to the axial length assumed by the Avanti system (23.95 mm).
      • Linderman R.
      • Salmon A.E.
      • Strampe M.
      • et al.
      Assessing the accuracy of foveal avascular zone measurements using optical coherence tomography angiography: segmentation and scaling.

      Statistical Analysis

      The objective was to investigate how foveal metrics differ between individuals with and without R402Q or S192Y, assuming other individual characteristics related to the metrics are similar. Furthermore, gene effects were defined as the differences in foveal metrics among participants with or without the R402Q or S192Y polymorphism. For each metric, Pearson median skewness was calculated to assess the distribution of the data. If the skewness was ≥ 0.4, a natural logarithmic transform was performed on the data and used for the following models. If the median skew was < 0.4, the raw data were used. To analyze the effect of R402Q and S192Y in our population, we used a linear mixed model to control for different demographic factors that have been shown to impact foveal morphology (R, version 4.0.3; R Foundation for Statistical Computing
      R Core Team
      R: A Language and Environment for Statistical Computing.
      ). The following factors were used as predictors: number of R402Q alleles, number of S192Y alleles, race, age, sex, and axial length. The S192Y and R402Q status were each listed as not having the polymorphism (0 polymorphism), heterozygous (1 polymorphism), or homozygous (2 polymorphisms). The following factors were outcomes: foveal pit depth, diameter, volume, and FAZ area. Once the initial model was created, nonsignificant variables were dropped (P > 0.05), and the model was rerun with only the significant variables in the model to acquire the final fit.

      Results

      Participant Characteristics

      There were 164 participants (67 male and 97 female), with a mean (± standard deviation [SD]) age of 30.5 ± 12.8 years. Self-reported ethnicity included 112 White participants, 3 American Indian/Alaskan native, 24 Asian, 1 native Hawaiian/Pacific Islander, 14 Black, 8 mixed-race participants, and 2 participants who did not report race. Across our participants, the average (± SD) foveal pit depth was 0.116 ± 0.021 mm, the average (± SD) foveal pit diameter was 1.936 ± 0.249 mm, the average (± SD) pit volume was 0.091 ± 0.036 mm3, and the average (± SD) FAZ area was 0.276 ± 0.111 mm2. Summarized quantitative data are shown in Figure 1.
      Figure thumbnail gr1
      Figure 1Comparisons of foveal and foveal avascular zone (FAZ) morphology metrics based on the TYR hypomorphic allele status. The average for each group is represented by the dashed line, the boxes represent the first to third quartiles, and the whiskers represent the minimum and maximum values for each group. Based on our mixed linear model, (A) pit depth, (B) pit diameter, (C) pit volume, and (D) FAZ area were significantly reduced in participants homozygous for R402Q. Participants heterozygous for R402Q had significantly smaller pit diameters and FAZ areas, where participants homozygous for S192Y had significantly smaller FAZ areas. Participants heterozygous for both R402Q and S192Y are included in both heterozygous categories for all metrics.

      TYR Genotype

      The average read depth for the analyzable target regions of TYR was 187.24 ± 84.97 (range, 40–578). Nine participants were homozygous for TYR R402Q, and 12 participants were homozygous for TYR S192Y. Forty-one participants were heterozygous for only R402Q, 25 participants were heterozygous for only S192Y, and 19 participants were heterozygous for both R402Q and S192Y. There were 58 participants with no copies of the R402Q or S192Y alleles. All 9 participants homozygous for R402Q (4 male, 5 female) and all 12 participants homozygous for S192Y (5 male, 7 female) identified themselves as White. The overall allele frequency for R402Q and S192Y was 23.8% and 20.7%, respectively. As both polymorphisms are known to have a higher allele frequency in people from Northern Europe, we further examined the frequency based on self-reported race. White participants in our study population had an allele frequency of 31.7% and 27.7% for R402Q and S192Y, respectively. Black individuals had an allele frequency of 3.5% (R402Q) and 7.1% (S192Y), whereas individuals from other racial backgrounds had an allele frequency of 8.1% (R402Q) and 4.1% (S192Y). These racial differences in allele frequency are consistent with previous studies.
      • Karczewski K.J.
      • Francioli L.C.
      • Tiao G.
      • et al.
      The mutational constraint spectrum quantified from variation in 141,456 humans.
      ,
      • Shriver M.D.
      • Parra E.J.
      • Dios S.
      • et al.
      Skin pigmentation, biogeographical ancestry and admixture mapping.

      Effect of R402Q and S192Y on the Foveal Region

      Genetic status and foveal morphology are summarized in Table 1. As both foveal volume and diameter had skewed distributions (Pearson median skew: 0.8 and 0.4, respectively), these data were transformed using the natural log, and the linear mixed model was run on the transformed data. Linear mixed model analysis used the following fixed variables: race, ethnicity, R402Q genotype, S192Y genotype, and sex. The continuous variables used were age and axial length.
      Table 1Summary of Foveal Morphology Metrics and FAZ Area Based on Genetic Status (Mean ± SD)
      TYR StatusDepth (mm)Diameter (mm)Volume (mm3)FAZ Area (mm2)
      No mutation0.119 ± 0.023 n = 582.015 ± 0.226 n = 580.100 ± 0.038 n = 580.321 ± 0.108 n = 56
      Heterozygous R402Q0.115 ± 0.022 n = 411.857 ± 0.203 n = 410.083 ± 0.032 n = 410.264 ± 0.112 n = 39
      Homozygous R402Q0.100 ± 0.028 n = 91.773 ± 0.280 n = 90.063 ± 0.026 n = 90.191 ± 0.105 n = 9
      Heterozygous S192Y0.117 ± 0.012 n = 251.983 ± 0.298 n = 250.099 ± 0.040 n = 250.273 ± 0.113 n = 24
      Homozygous S192Y0.119 ± 0.010 n = 121.900 ± 0.267 n = 120.085 ± 0.027 n = 120.244 ± 0.053 n = 11
      Heterozygous

      R402Q and S192Y
      0.109 ± 0.022 n = 191.901 ± 0.245 n = 190.084 ± 0.029 n = 190.234 ± 0.098 n = 19
      All participants0.116 ± 0.021 n = 1641.936 ± 0.249 n = 1640.091 ± 0.036 n = 1640.276 ± 0.111 n =158
      FAZ = foveal avascular zone; SD = standard deviation.
      Results showed significant reductions in all foveal and FAZ metrics for those homozygous for R402Q. There were significant reductions in diameter and FAZ area in those heterozygous for R402Q, whereas those homozygous for S192Y had significantly decreased FAZ areas. Those heterozygous for S192Y had no significant difference in foveal or FAZ morphology (Table S1, available at www.ophthalmologyscience.org). Examples of the subjects with the smallest and largest foveal metrics and FAZ areas are shown in Figure 2. Black race was associated with larger pit volumes (P = 0.038), diameters (P = 0.045), and larger FAZ areas (P = 0.031). White race, Asian race, and younger age were associated with smaller FAZ areas (P = 0.0005, P = 0.015, P = 0.0047, respectively). Longer axial length was associated with smaller pit depths (P = 0.0001) and smaller pit volumes (P = 0.0029), whereas male sex was associated with smaller FAZ areas (P = 0.0004). Predictors and associations for our complete model are summarized in Table S1 (available at www.ophthalmologyscience.org).
      Figure thumbnail gr2
      Figure 2Images and measurements from eyes with the smallest and largest foveae based on a composite ranking. To create the composite ranking, each eye was rank ordered for each of the 4 metrics (pit depth, pit diameter, pit volume, and FAZ area), and their total rank was calculated by summing their ranks from each metric. The eyes with the lowest composite score were deemed to have the smallest fovea (top rows) and those with the highest composite score were deemed to have the largest fovea (bottom rows). For each participant, their OCT B-scan, macular thickness map, and averaged OCT angiography images are shown along with their measurements of pit depth, diameter, volume, and FAZ area. FAZ = foveal avascular zone.
      Using only the significantly associated variables (Table 2) for pit depth, linear mixed model analysis showed that individuals homozygous for TYR R402Q had pits that were 0.020 mm shallower than those without R402Q polymorphism (P = 0.007). Likewise, we found that the individuals homozygous for TYR R402Q had pits that were reduced in volume by 35% in comparison with those without the R402Q polymorphism (P = 0.0005). The analysis for pit diameter showed individuals heterozygous for TYR R402Q had diameters reduced by 5%, whereas participants homozygous for TYR R402Q had diameters reduced by 10% in comparison with those with no R402Q alleles (P = 0.0094 and P = 0.0052, respectively). Compared with those with no R402Q alleles, individuals heterozygous for TYR R402Q had FAZ areas that were 0.036 mm2 smaller, while those homozygous for TYR R402Q had FAZ areas that were 0.111 mm2 smaller (P = 0.025 and P = 0.0012, respectively). Finally, subjects homozygous for TYR S192Y also had reduced FAZ areas by 0.038 mm2 compared with those with no TYR S192Y mutations (P = 0.016).
      Table 2Effect Size of the Predictors Found to Be Significant in the Final Linear Regression Model
      PredictorsDepth (mm)Diameter (%)
      Data were transformed using the natural log; effect size is reported as a percentage.
      Volume (%)
      Data were transformed using the natural log; effect size is reported as a percentage.
      FAZ Area (mm2)
      Effect SizePEffect SizePEffect SizePEffect SizeP
      Heterozygous R402QNS−4.790.0094NS−0.0360.025
      Homozygous R402Q−0.0200.007−9.950.0052−34.700.0005−0.1110.0012
      Heterozygous S192YNSNSNSNS
      Homozygous S192YNSNSNS−0.0380.016
      MaleNSNSNS−0.0600.0004
      White raceNSNSNS−0.0590.0005
      Asian raceNSNSNS−0.0200.015
      Black raceNS11.060.002834.040.00220.0910.032
      AgeNSNSNS0.0020.0049
      Axial length (mm)−0.00580.0001NS−6.930.003NS
      FAZ = foveal avascular zone; NS = not significant (in final linear mixed effects regression model); – = no significant effect.
      Data were transformed using the natural log; effect size is reported as a percentage.

      Discussion

      This study examined the association between TYR R402Q and S192Y hypomorphic alleles and foveal structure. Compared with participants with no R402Q or S192Y alleles, those heterozygous for the TYR R402Q hypomorphic allele had significantly smaller foveal diameters (7% decrease on average) and FAZ areas (21% decrease on average). Participants homozygous for R402Q showed even larger effects, with a 16% reduction in pit depth, 12% reduction in foveal diameter, 37% reduction in foveal volume, and 41% reduction in FAZ area compared with those with no R402Q or S192Y alleles. Only the FAZ area was significantly reduced in subjects homozygous for TYR S192Y (by 24% on average). These effects in the raw data were generally consistent with the effects observed using the linear mixed model analysis.
      Additional support for our findings comes from a recent genome-wide association study of inner retinal morphology using the UK Biobank.
      • Currant H.
      • Hysi P.
      • Fitzgerald T.W.
      • et al.
      Genetic variation affects morphological retinal phenotypes extracted from UK Biobank optical coherence tomography images.
      The authors found that the S192Y variant was associated with increased retinal thickness (i.e., a shallower foveal pit), whereas R402Q was not associated with retinal thickness in their cohort. The different metrics used in Currant et al
      • Currant H.
      • Hysi P.
      • Fitzgerald T.W.
      • et al.
      Genetic variation affects morphological retinal phenotypes extracted from UK Biobank optical coherence tomography images.
      may explain the contrasting results observed in our study. That study assessed the average retinal nerve fiber layer thickness and GCL-IPL layer thickness across the Macula6 grid and the total retinal thickness at the fovea, whereas our study assessed the shape of the foveal pit and the FAZ area (using OCT angiography). Although our results from the full model analysis show the same overall trend as our condensed analysis using the model with significant variables, the full model showed that the S192Y variant just missed statistical significance in its association with FAZ area in the heterozygous state (P = 0.052) and was significant in the homozygous state (P = 0.015) state (Tables S1 and S2, available at www.ophthalmologyscience.org). Nevertheless, both studies support a role for tyrosinase activity in the development of the size and shape of the fovea, though further work examining the contribution of the S192Y and R402Q alleles is warranted (especially related to any additive effects they may have when present in cis vs. trans).
      There are important limitations to this study. First, there were a relatively small number of participants who were homozygous for R402Q (n = 9) or S192Y (n = 12). This may also explain the differences in results when we compare the results here with those of Currant et al.
      • Currant H.
      • Hysi P.
      • Fitzgerald T.W.
      • et al.
      Genetic variation affects morphological retinal phenotypes extracted from UK Biobank optical coherence tomography images.
      Furthermore, 10 children aged less than 16 years were included in this study, which could potentially confound metrics of pit morphology for their respective genetic group (since foveal development may continue after birth
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      • et al.
      Maturation of the human fovea: correlation of spectral-domain optical coherence tomography findings with histology.
      ). However, the statistical analyses accounted for foveal differences due to age when examining the effects of the TYR hypomorphic alleles. In addition, although previous research has shown that these alleles can result in decreased melanin in the retina, choroid, iris, skin, or hair,
      • Dolinska M.B.
      • Kus N.J.
      • Farney S.K.
      • et al.
      Oculocutaneous albinism type 1: Link between mutations, tyrosinase conformational stability, and enzymatic activity.
      • Tripathi R.K.
      • Giebel L.B.
      • Strunk K.M.
      • Spritz R.A.
      A polymorphism of the human tyrosinase gene is associated with temperature-sensitive enzymatic activity.
      • Chaki M.
      • Sengupta M.
      • Mondal M.
      • et al.
      Molecular and functional studies of tyrosinase variants among Indian oculocutaneous albinism type 1 patients.
      we did not do any qualitative or quantitative assessment of melanin in our participants. We also did not account for any potential environmental factors that may impact retinal development of these individuals. Finally, we examined only 2 hypomorphic variants on the TYR gene. Other hypomorphic alleles in the TYR and OCA2 genes have been reported.
      • Norman C.S.
      • O'Gorman L.
      • Gibson J.
      • et al.
      Identification of a functionally significant tri-allelic genotype in the Tyrosinase gene (TYR) causing hypomorphic oculocutaneous albinism (OCA1B).
      ,
      • Dolinska M.B.
      • Kus N.J.
      • Farney S.K.
      • et al.
      Oculocutaneous albinism type 1: Link between mutations, tyrosinase conformational stability, and enzymatic activity.
      ,
      • Dolinska M.B.
      • Kovaleva E.
      • Backlund P.
      • et al.
      Albinism-causing mutations in recombinant human tyrosinase alter intrinsic enzymatic activity.
      • Simeonov D.R.
      • Wang X.
      • Wang C.
      • et al.
      DNA variations in oculocutaneous albinism: an updated mutation list and current outstanding issues in molecular diagnostics.
      • Andersen J.D.
      • Pietroni C.
      • Johansen P.
      • et al.
      Importance of nonsynonymous OCA2 variants in human eye color prediction.
      Further assessment of how these alleles impact foveal morphology, as well as continued identification of other potential hypomorphic alleles in genes implicated in the pigmentation pathway, would be the next steps in understanding the large variability of normal foveal anatomy.
      In conclusion, the results presented expand our understanding of the normal variability in foveal morphology. On one end of the pigmentation spectrum are Black individuals, who have deeper and broader foveal pits.
      • Wagner-Schuman M.
      • Dubis A.M.
      • Nordgren R.N.
      • et al.
      Race- and sex-related differences in retinal thickness and foveal pit morphology.
      ,
      • Zouache M.A.
      • Silvestri G.
      • Amoaku W.M.
      • et al.
      Comparison of the morphology of the foveal pit between African and Caucasian populations.
      On the other end are patients with albinism, who range from having an absent foveal pit to a shallow pit.
      • Thomas M.G.
      • Kumar A.
      • Mohammad S.
      • et al.
      Structural grading of foveal hypoplasia using spectral-domain optical coherence tomography: a predictor of visual acuity?.
      White individuals and individuals from other racial backgrounds fall in between these extremes. There are likely other genes that regulate the formation of the foveal pit; however, our data suggest that the variations we see within racial groups may be driven (at least in part) by hypomorphic alleles in TYR.

      Acknowledgments

      The authors thank Erin Curran, Brian Higgins, Phyllis Summerfelt, and Maureen Tuffnell for their contributions to this work.

      Supplementary Data

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