Heritability of Macular Thickness Determined by Optical Coherence Tomography

Abstract

purpose. To examine whether genetic factors significantly influence macular thickness in healthy older subjects.

methods. A classic twin study was performed to compare the correlation of macular thickness between monozygotic (MZ) and dizygotic (DZ) twins in a sample of population-based volunteer twins. The study included 109 white twin pairs from 50 to 80 years of age without evidence of manifest eye disease and corrected visual acuity better than 6/7.5. Dilated macular optical coherence tomography (OCT), fundus photography, clinical examination, ocular biometry, a health-dietary questionnaire, and subjective autorefraction were performed on all subjects.

results. Correlation of retinal thickness was significantly greater between MZ twin pairs than DZ pairs in all macular regions. The MZ-to-DZ correlation was 0.88:0.44 for the foveal region, 0.79:0.47 for the inner macular region, and 0.81:0.50 for the outer macular region. With adjustment for significant covariates and model fitting, final heritability estimates of 85%, 81%, and 81%, respectively, were obtained. A significant correlation between foveal thickness and gender was present, with the men having significantly thicker foveae. There was a significant negative correlation between outer macular thickness and axial length.

conclusions. This study confirms that macular thickness in older healthy subjects, as measured by OCT, may be affected by genetic factors. Factors such as axial length, gender and age, warrant further examination in larger population-based studies, as variables that may influence macular thickness. This finding suggests an inherited basis of macular thickness and may help in the understanding of the factors that govern macular structure and function.

Accurate and reproducible in vivo measurement of retinal thickness has only recently become possible with the advent of technology such as scanning laser polarimetry, retinal tomography (Heidelberg Retinal Tomograph [HRT]; Heidelberg Engineering, Heidelberg, Germany), and optical coherence tomography (OCT). ¹ Clinically applicable uses of OCT retinal thickness scanning include a potential role in the diagnosis and management of glaucoma, via examination of retinal nerve fiber layer thickness (RNFLT). ^{2 3 4} Similarly, OCT has been used in diagnosis and treatment of vitreoretinal disorders, and monitoring of retinal thickness during treatment of conditions such as diabetic macular edema. ⁵ Previous studies in animals have indicated that structures identified ex vivo by histology correlate with OCT determination of retinal substructure, validating the utility of this technique. ^{6 7}  

The finding that a trait has a high genetic component in the normal population is very important if that trait can be linked to or is an independent subphenotypic contributor to disease. It is these specific traits with a genetic basis that will allow more effective gene searching, ⁸ through linkage and association studies, to identify genes involved in complex diseases such as AMD and glaucoma. As a consequence, we undertook this study to assess what role genetic factors have in the determination of macular topography in subjects without eye disease, with the idea that learning more about the healthy macula may allow us to determine better how disease influences traits. 

Twins offer an ideal opportunity to examine the contribution made to both genetic and environmental factors that influence a trait. As such, classic twin studies are used to establish the genetic basis of a trait by comparing correlations between identical (monozygotic; MZ) and nonidentical (dizygotic; DZ) twins. A significantly greater correlation in MZ than DZ twins indicates a role for genetic factors. In one recent twin study, investigators looked at the peripapillary RNFLT and noted that it is predominantly determined by genetic factors. ⁹ They noted a heritability of 78% for nerve fiber layer (NFL) thickness. As a consequence, we wanted to use OCT as a tool to assess macular thickness in individuals with healthy eyes. 

Materials and Methods

Subjects

All MZ and DZ twin pairs were recruited through the National Health and Medical Research Council (NHMRC)-funded Australian Twin Registry (ATR) as part of an eye study of older twins. All individuals involved underwent a full eye examination. Inclusion in this analysis required best-corrected visual acuity of 6/9 or better. Twins with a history of glaucoma, diabetes, ¹⁰ retinal disease, or visually significant cataract (defined by a grading on the Lens Opacities Classification System II system) ¹¹ were excluded from the analysis. 

Zygosity

Twin zygosity was determined by asking a standardized and validated set of questions used by the Australian Twin Registry (ATR). ^{12 13} These questions have been shown to be 95% accurate in the correct classification of zygosity. ¹⁴ Approximately 30% of participants had had their zygosity status confirmed through prior DNA testing from previous twin studies. Twins in which there was a failure to resolve the question of zygosity had a zygosity test performed. Standard zygosity testing included 11 highly polymorphic and heterozygotic markers (SEfiler; Applied Biosystems, Inc., Foster City, CA). Zygosity testing was necessary in only two of the twin pairs in this cohort. 

Examination

All test measurements were performed on both eyes. Subjects initially provided subjective refraction and a medical-dietary history, before undergoing pupillary dilation with 1.0% tropicamide. Objective refraction and biometry measurements followed pupillary dilation. The testing procedures included autorefraction (model KR 8100 autorefractor; Device Technologies, Mulgrave, Australia) and A-scan (IOL Master; Carl Zeiss Meditec, Inc., Dublin, CA) and provided biometric measurements of axial length, anterior chamber depth, and keratometry readings and clinical examination when a slit lamp was used to assess the presence of cataracts, and, if present, these were graded according to the Lens Opacities Classification System II. Details of OCT and fundus photography are provided in the following sections. 

All twins gave informed consent before participating in the project according to the Declaration of Helsinki, and the study was approved by the Royal Victorian Eye and Ear Hospital (RVEEH) Human Research and Ethics Committee. 

Optical Coherence Tomography

The macular region was examined (OCTStratus 3; Carl Zeiss Meditec, Inc., Dublin, CA) using the fast macular scan mode. The patient was asked to fixate on a central light while the examination was performed. The fast macular scan mode acquired six 6-mm radial line scans within 1.92 seconds of scanning, with 768 A-scans obtained in total. No allowance was made for axial length or refraction of participants at this stage of testing. Scans that were identified by the operator as having a low signal-to-noise (SNR) ratio, misidentification of the retinal limits, or fixation loss were repeated. Later review of the six individual scans in cross-sectional mode identified a further 14 scans that were excluded from analysis, with evidence of artifact (fixation error; n = 4), misidentification of retinal layers (n = 7), and low signal-to-noise-ratio (n = 3). 

The OCT was analyzed by using the retinal thickness tabular output mode provided by the manufacturer. This mode averages the retinal thickness at three map diameters: 1.00-, 3.00-, and 6.00-mm eccentricity from fixation, as defined by the OCT software (Stratus OCT version 3.0.1; Carl Zeiss Meditec, Inc.). These areas will subsequently be referred to as the foveal, inner macular, and outer macular regions, respectively, as denoted by the OCT. The inner and outer macular regions were further divided into temporal, superior, nasal, and inferior components by the standard system software. The mean retinal thickness was calculated for each of these nine regions and judged as the distance between the two highly refractile surfaces: anteriorly the inner limiting membrane (ILM) and posteriorly the junction of the photoreceptor inner and outer segments. 

Fundus Photography

Color fundus photographs including both 35° stereo macular photographs and optic nerve head were taken with a retinal camera (TRC 50X; Topcon, Tokyo, Japan). The photographs were reviewed by two independent observers who graded for the presence of any macular disease. Any eyes graded as having either early or late AMD, as defined by the International Classification System were excluded from the analysis. ¹⁵ The right eye was chosen for the analysis, except in 13 cases in which unilateral disease was present, and the left eye was substituted: early AMD (n = 10), epiretinal membrane (n = 2), and central serous retinopathy (n = 1). Seventeen pairs were excluded from this analysis as either one or both subjects had signs of AMD bilaterally. 

Statistical Analysis

The classic twin study considers that the variance of a phenotype can be due to a combination of genetic and environmental factors. Classic twin models are typically modeled with the assumption that the residual variance (ς²) can be partitioned into three components: ς_A ² representing additive genetic factors, ς_C ² representing common environmental factors within the same pair, and ς_E ² representing individual environmental factors. Because MZ twins share all their genetic material, and DZ twins share, on average, half of their genes, covariance for MZ twins can be defined as ς_A ² + ς_C ², whereas the covariance for DZ twins is defined as 0.5ς_A ² + ς_C ². The classic twin model further assumes that MZ and DZ twins share a common family environment—the equal environment assumption. ¹⁶ The heritability (h²), or proportion of residual variance attributed to additive genetic factors, is the ratio of additive genetic variance to residual variance (ς_A ²/ς²). 

The Fisher statistical package was used to fit all models according to maximum likelihood ¹⁷ and to test the assumptions of the models. ¹⁸ Modeling was based on continuous, normally distributed variables in all cases. Statistical inference and the choice of parsimonious models were based on asymptotic likelihood theory and the information criterion of Akaike. ¹⁹  

Results

Demographic Characteristics

This analysis includes a subset of participants involved in the twin eye study (n = 300), inclusive of participants examined between March and December 2004, and exclusive of conditions noted in the Methods section. A total of 109 twin pairs (58 MZ and 51 DZ pairs) were included in the analysis, 148 (67.9%) women and 70 (32.1%) men. Baseline characteristics of both MZ and DZ twins are summarized in Table 1 . There was a significant difference in the mean age of the MZ twin pairs (57.6 years) compared with the DZ twin pairs (59.7 years; P = 0.007), and also a significantly higher proportion of women in the DZ group (P = 0.026). There was a larger proportion of women in both groups of twins, this phenomenon being well recognized in twin studies. The mean axial length was longer in the MZ group (23.58 mm) than in the DZ group (23.33 mm), although the difference did not reach statistical significance (P = 0.08). There was no significant difference between the groups with reference to BMI (P = 0.94). 

Macular Thickness

Fovea.

The mean foveal thickness across the study population was 210.3 ± 21.1 μm, with no difference between MZ (210.9 ± 22.6 μm) and DZ (209.5± 19.4 μm; P = 0.63) twins. The only factor that impacted significantly on foveal thickness was gender (r = −0.333; P < 0.001), which explained 11% of the variance in foveal thickness in this population—the male gender being associated with increased foveal thickness. Neither age (P = 0.245) nor axial length (P = 0.407) was significantly associated with mean foveal thickness. Body mass index (BMI) and smoking were not significantly associated with macular thickness in any region after multivariate analysis. 

Inner and Outer Macula.

The mean macular thickness in the inner and outer macula was calculated by averaging the superior, nasal, inferior, and temporal readings. The mean inner macular thickness was 272.2 ± 16.2 μm, and the mean outer macular thickness was 235.1± 15.6 μm. There was no difference in either the mean inner or outer macular thickness between MZ and DZ twins (P = 0.77, 0.10, respectively). The only factor significantly associated with mean inner macular thickness was age (r = −0.13; P = 0.04), which explained only 2% of trait variance. Age was associated with decreased inner macular thickness in this cohort. Both axial length (r = −0.21; P = 0.002) and age (r = −0.23; P = 0.001) were associated with outer macular thickness, and both explained approximately 5% of trait variance in the population. Increasing axial length and age were associated with decreased outer macular thickness. Gender was not significantly associated with either mean inner or outer macular thickness. 

Heritability Estimation

The nonadjusted intrapair correlations are presented in Figure 1in scatterplots. The pair-wise correlations for the foveal region are 0.88 for MZ twin pairs and 0.44 for DZ twin pairs. The correlations for the inner macula region are 0.79 and 0.47 for MZ and DZ pairs respectively, whereas the correlations for the outer macular regions are 0.81 and 0.50, respectively. Crude heritability estimates can be calculated as twice the difference between the MZ and DZ correlations and were estimated as 87%, 64%, and 61%, respectively, for the foveal, inner macular, and outer macular regions. 

The Fisher statistical program was used to model variance into three components: additive genetic (A), common environmental (C), and unique environmental (E) variance. All statistical comparisons are made in reference to the full ACE model. There were no statistically significant differences between the AE and ACE models in all three regions (Table 2) . In all cases, the CE model was a significantly worse fit than either the AE or ACE model (P < 0.001; Table 2 ), indicating that there was little role for the common environment in influencing macular thickness in this cohort of older twins. The AE model was chosen as the best fitting model, because it was the most parsimonious (fewest latent variables) and was not significantly different from the full ACE model. There was no evidence for any dominant effect on macular thickness within any region of the macula examined. The model fitting estimates as shown in Table 3are all adjusted for significant covariates such as age, gender and axial length. In all regions, the adjusted heritability estimates were greater than 80%. Final estimates for heritability of foveal thickness were 85% (77%–92%); for inner macular thickness, 81% (73%–89%); and for outer macular thickness 81% (74%–89%). Heritability estimates were also calculated for each individual quadrant (superior, nasal, inferior, and temporal) within the inner and outer macula regions. These estimates were not significantly different from that of the mean thickness and are therefore are not shown. 

Discussion

This study provides evidence to support the notion that genetic factors play a role in determining macular thickness out to 3 mm from the fovea, as measured by optical coherence tomography (OCT) in older subjects without eye disease. This finding also appears to be consistent across all regions of the macula, even where there is considerable variation in retinal thickness. The findings reported herein build on findings reported in a recent twin study of peripapillary RNFLT, ⁹ in which a high degree of genetic control was also found, with an age-adjusted heritability of 78%. In light of the increasing use of OCT technology for examination of retinal thickness and structure in the investigation and observation of ophthalmic diseases, this finding is very significant. 

Knowledge of the factors that govern the structure and morphology of the macula, as measured by OCT, are still in their infancy. Variation in retinal thickness across the macula is well defined, with thickness in the peri- and parafoveal regions appearing to diminish with age, although the foveola appears to be spared from this phenomenon. ²⁰ Previous studies on the effect of axial length on macular thickness have shown variable results. Axial length of the eye had not been found to be a significant influence on retinal thickness, ²¹ even with correction for theoretical magnification effects in higher nonpathologic myopes. ²² However two more recent studies demonstrated a relationship between a longer axial length and a thicker central retina in Asian populations. ^{23 24} Gender and body mass index (BMI) have also been implicated as significant factors in retinal thickness. ²³  

The adjusted heritability in the present study was consistently high in all macular regions examined: 85%, 81%, and 81%, respectively, across the foveal, inner macular, and outer macular regions after adjustment for significant covariates, as previously noted. Breakdown of these regions into superior, nasal, inferior, and temporal regions of the inner and outer macula, also showed that heritability remained consistent with the mean of its region, despite local variation in thickness. The high degree of heritability of macular thickness noted in this study suggests that genetic factors govern both the structure and organization of the macula. Variance component modeling further suggested that the most parsimonious model included components only for additive genetic variance and nonshared environmental variance only. There was no apparent role for the common environment in influencing macular thickness, although the sample size is unlikely to be large enough to detect common environmental effects. 

Our study allowed exploration of common biological variables that influence macular thickness among the normal population. We noted a gender-related variation of retinal thickness in the foveal region that accounted for 11% of all trait variance in the foveal region. This effect was most pronounced in the men, in whom a thicker fovea was present than in the women. However, this finding was not present in the inner and outer macula, indicating that any sex-specific effects on retinal thickness were confined to the foveal region. Beyond 1.5 mm from the central fovea, it was noted that axial length became a significant covariate. This negative correlation implied that retinal thickness in this region was reduced in subjects with longer axial lengths. This association was not present beyond 1.5 mm from the fovea, supporting the findings of Wakitani et al. ²² who also noted no influence of axial length on the central 3.45-mm region, even after correction for theoretical retrograde magnification. In other studies, where no association between axial length and macular thickness has been found, it should be noted that in our population, minimal refractive error was present in comparison to some of the Asian studies. 

We found evidence of a significant negative correlation between age and retinal thickness that was present only between a 0.5- and 3-mm diameter from the fovea. This was not so for the central 1 mm of the macula, implying that there was an age-related decrease in retinal thickness away from the fovea, in agreement with the findings of Kanai et al. ²⁰ This phenomenon of age-related loss of macular thickness in the nonfoveal macula is well documented in other studies such as in peripapillary NFL thickness, where this thinning appears to occur as a secondary event to a loss of NFL axons. 

The limitations of this study include the limitations of a commercially available OCT scanner. The major flaw with current software is that the algorithms are unable to differentiate reliably between the junction of the photoreceptor inner and outer segments and the RPE–choriocapillaris interface. ²⁵ Hence macular thickness as measured by OCT may not equate with true anatomic thickness, as the outer photoreceptor segments may be excluded. This will consequently lead to underestimation of true macular thickness. Further bias may have been introduced by the use of the fast macular scan, as the duration scanning is increased and the possibility of artifact is increased. ²⁶ However all attempts to eliminate artifact were made before analysis, and it should be noted that the presence of random error in the analysis would lead only to underestimation of the genetic effect. ²⁷  

Many retinal diseases primarily affect the macula, yet there is still incomplete understanding of why the macula is more vulnerable to degenerative change than other regions of the retina. A key question to be answered is what are normal age-related macular anatomic changes and what are those consistent with disease. We already have some knowledge of normal age-related structural changes in the macula, in particular changes in the morphology of RPE cells in the macula region ^{28 29} ; declining rod density, most pronounced at 5 to 8 mm from the fovea ³⁰ ; and increasing accumulation of cholesterol within Bruch’s membrane. ³¹ By understanding more about the natural biology of the aging macula and what roles environmental and genetic factors play, we may in the future be better able to target genetic research. This study suggests that human macular thickness is controlled, at least to some degree, by genetic factors. As the resolution of the OCT modality further improves, it would be worthwhile to examine individual retinal sublayers and note the relative genetic and environmental influence on natural biological variation in an aging population. 

In conclusion, we found that the thickness of the human macula, as measured by OCT, is determined at least to some degree by genetic factors. This finding is supported by the variance component models that were fitted for each region of the macula. There was also a role for gender, age, and axial length in determining macular thickness. Biometric factors that influence macular thickness warrant further investigation and should be taken into account in ongoing research of the macula using the OCT modality. The exposition of retinal subphenotypes that are specifically influenced by genetics, time, or the environment will help us answer questions about the normal aging process as well as disease. 

 Supported by the Ophthalmic Research Institute of Australia (ORIA). MDC is the recipient of a National Health and Medical Research Council (NHMRC) postgraduate scholarship.

 Submitted for publication May 16, 2005; revised August 14, 2005; accepted November 28, 2005.

 Disclosure: M. Chamberlain, None; R. Guymer, None; M. Dirani, None; J. Hopper, None; P. Baird, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Table 1.

 

Demographic Characteristics of Participant Twin Pairs by Zygosity

	MZ Twin Pairs	DZ Twin Pairs	P
Twin pairs (n)	58	51	—
Gender (male/female)	44/72	26/76	0.026
Age (y)	57.6	59.7	0.007
Axial length (mm)	23.58	23.33	0.08
BMI	25.65	25.67	0.94
Foveal thickness (μm)	210.9	209.5	0.63
Inner macula thickness (μm)	272.5	271.8	0.77
Outer macula thickness (μm)	236.7	232.3	0.10

Figure 1.

 

Intrapair correlations for foveal, inner macular, and outer macular thickness in MZ and DZ twin pairs.

Table 2.

 

Results of ACE Model-Fitting with Inclusion of Significant Covariates

Measure	Model	AIC	Log-Likelihood	ΔLog-Likelihood	df	P
Fovea	ACE	1438.75	−719.377
	AE	1439.45	−719.726	0.349	1	0.404
	CE	1456.79	−728.395	9.018	1	<0.001
Inner macula	ACE	1287.91	−643.955
	AE	1290.15	−645.074	1.119	1	0.135
	CE	1310.75	−655.377	11.422	1	<0.001
Outer macula	ACE	1307.80	−653.901
	AE	1310.75	−655.377	1.476	1	0.086
	CE	1320.31	−660.157	6.256	1	<0.001

Table 3.

 

Heritability Estimates with Adjustment for Significant Covariates

Trait	Heritability (%)	95% CI
Fovea	85	77–92
Inner macula	81	73–89
Outer macula	81	74–89

The authors thank the Australian Twin Study for access to the Twin Register.