Abstract
Research into the genetic effects and specific genes associated with retinal vascular caliber, a risk marker of cardiovascular diseases, may provide new insights into the genetic contribution of early microvascular disease. A combined 374 monozygotic and 536 dizygotic twin pairs and 322 siblings from the Twins Eye Study in Tasmania (TEST) and the Brisbane Adolescent Twin Study (BATS) underwent complete ophthalmic examinations including retinal photography, and bilateral retinal vascular caliber was measured. Structural equation modeling was used to estimate the heritability. Genome-wide linkage analysis was conducted on 836 individuals from 381 BATS families with adjustments for age, sex, and other covariates. The heritability for retinal arteriolar caliber was 59.4% (95% confidence interval, 53.2% to 64.7%) and 56.5% (50.1% to 61.9%) in TEST and BATS, respectively, and for venular caliber was 61.7% (55.6% to 67.0%) and 64.2% (58.7% to 68.8%), after adjusting for age, sex, and body mass index. Two multipoint peaks detected on chromosomes 3p12.3 and 8p23.1 for retinal arteriolar caliber had suggestive linkage, with the highest multipoint peak logarithm of odds (LOD) score of 2.24 on chromosome 8p23.1 (genome-wide P-value=7.0×10−4). Two suggestive LOD scores for venular caliber were identified on chromosomes 2p14 and 9q21.13. The largest multipoint LOD score was 2.69 on chromosome 2p14 (genome-wide P-value=2.0×10−4). In this large twin population, genetic factors appear to play a significant role in the variation of retinal vascular caliber. Several putative loci were identified for retinal vascular caliber.
Keywords: twins, heritability, retinal vascular caliber, genome-wide linkage analysis, genetics
Introduction
Recent population studies suggest that a quantitative assessment of retinal vascular caliber may allow understanding of early structural changes in the microcirculation, and their relationship to risk of hypertension, diabetes and cardiovascular disease.1–5 Variation in retinal arteriolar and venular caliber appear to reflect differential effects of systemic, environmental, and possibly genetic factors.1 For example, narrower retinal arterioles are associated with higher levels of past, current, and future blood pressure and obesity, and predict the incidence of diabetes and coronary heart disease.6–12 In contrast, wider retinal venules are associated with impaired fasting glucose metabolism, dyslipidemia, obesity, inflammation, endothelial dysfunction and cigarette smoking, and predict the risk of stroke and coronary heart disease.13–18
Data from a familial aggregation study,19 a twin study20 and a genome-wide linkage study21 suggest that genetic factors contribute substantially to the normal variation in retinal vascular caliber. Several putative loci have also been identified.21 Research into the genetic effects and specific genes associated with retinal vascular caliber may provide new insights into the genetic contribution of early microvascular disease.1,22,23
In the current study, we examined the heritability of retinal vascular caliber in a sample of Australian twins age ranged 5 to 90 years. We then performed a genome-wide linkage scan in a subsample of 836 individuals from 381 families to identify underlying quantitative trait loci (QTL).
Methods
Study Population
The study populations were derived from predominately Caucasian twins, recruited from the Twins Eye Study in Tasmania (TEST), which included twins from the Tasmanian Infant Health Cohort and the Australia Twin Registry,24 and the Brisbane Adolescent Twin Study (BATS), details of which have been previously published.25,26 We invited all twins and their non-twin siblings to participate in an extensive eye examination and complete a detailed questionnaire providing relevant sociodemographic and medical information. Participants from both study sites were examined by the same research team following the same protocol.
The study was approved by the ethics committees of the Royal Victorian Eye and Ear Hospital, the Royal Hobart Hospital, and the Queensland Institute of Medical Research as well as the Australian Twin Registry, and adhered to the tenets of the Declaration of Helsinki. Written informed consent, including consent for genetic analyses, was obtained from all participants or their legal guardians, with the participants’ assent prior to examination.
There were 2,210 participants from 1,040 families in the total sample (1,021 from TEST and 1,189 from BATS). Of these, 2,091 persons had retinal vascular caliber data for both right and left eyes, and an additional 51 individuals had either right or left eye retinal vascular caliber data, comprising 374 monozygotic (MZ) and 536 dizygotic (DZ, 256 opposite sex DZ) twin pairs as well as 322 participants analyzed as singletons in the heritability analysis. Data were analyzed by study site because of potential differences in population characteristics.
Retinal Photography and Grading
All twins and non-twin siblings had 10° stereoscopic optic disc-centered photographs using a Nidek 3-Dx/F fundus camera (Nidek, Gamagori, Japan) after dilatation of the pupils with tropicamide 1% or cyclopentolate 1%. After digitalization of photographs, retinal vascular caliber was measured with a computer-assisted software (IVAN, University of Wisconsin) according to a standardized protocol.27 Two trained graders, masked to participant characteristics, performed the vessel measurements on the optic disc-centered image for both eyes for all participants. The largest six arterioles and venules coursing through a zone between half to one disc-diameter from the optic disc margin were measured. Images were considered ungradable if quality was poor or if the largest six vessels could not be measured. Estimates were summarized as central retinal arteriolar equivalent (CRAE) and central retinal venular equivalent (CRVE) representing the average diameter of arterioles and venules of the eye, respectively, using a revised Knudtson-Parr-Hubbard formulae.28 Reliability of the retinal vessel measurement has been published elsewhere.25 The intra-grader variation was assessed in 67 randomly selected retinal photographs. Intra-grader intraclass correlation coefficient was 0.95 for CRAE and 0.99 for CRVE, and inter-grader reliability was assessed in 52 randomly selected retinal images, and interclass correlation coefficient was 0.93 for CRAE and 0.98 for CRVE.
Zygosity Testing and Genotyping Information
Genomic DNA was extracted from either buccal swabs or venous blood samples. Zygosity in twins of the same sex was confirmed by genotyping up to twelve highly polymorphisms short tandem repeat (STR) markers.29
The STR Genome Scan
The genome scan for the BATS was performed at three different laboratories: the Australian Genome Research Facility (AGRF, Melbourne, Australia), Center for Inherited Disease Research (CIDR, Baltimore, Maryland), and Marshfield Mammalian Genotyping Service (MARS, Marshfield, Wisconsin). Preliminary data of the genome scan has been described in a previous analysis.30 In summary, 644 families comprising 2,756 individuals, including 1,199 parents, had whole genome scan in the BATS (an average of 410 microsatellite markers from AGRF, 386 markers from CIDR, and 394 markers from MARS). After passing our quality-control standards, there were 1,190 microsatellite markers (900 unique markers) included in the final analysis; the genome-wide average marker distance for sib pairs both typed was 8.31 centimorgans (cM). Currently, there are no genome-wide scans performed for the TEST cohort.
The Single Nucleotide Polymorphisms (SNP) Genome Scan
The 100K SNP data were from the Affymetrix 100K SNP chip marker set (109,511 SNPs) genotyped at the AGRF. All DNA of children for 169 families of BATS were genotyped. Genotyping data (50K Array Xba 240) were also available for both parents in 104 families, and one parent in 7 families, with 58 families having no parent genome information. We used the program GRR to confirm the expected family relationship between individuals.31 After removing approximately 5% SNPs according to Hardy-Weinberg equilibrium testing (dropping cutoff P-value <0.001), there were 6.1% SNPs with minor allele frequency (MAF) < 1% in the SNP scan set. In total, there were 109,511 SNPs in the scan set and 102,802 of the SNPs (93.9%) were common and SNPs with MAF < 5% were excluded from the SNP linkage analysis. Among these families, 192 individuals from 90 families had retinal vascular caliber measurements.
Combined STR and SNP Linkage Set
A smaller linkage data set (15K) was created from the 100K SNP scan dataset by restricting the minimum distance between adjacent SNPs to be 0.1cM. The additional 169 families with genome scan of SNPs data were then combined with the families with STR to increase sample size and genotyping information, and thus to increase statistical power to detect QTL.
The pedigree information for combined linkage set (all STR markers and linkage SNP set) was from 811 families consisting of 1,409 parents and 1,946 children. Taken together, there were 17,006 markers (STR and SNP) and the average marker distance was 0.21 cM with the total coverage of 3,570 cM.
Definition of Other Variables
In brief, all twins and siblings completed a detailed questionnaire including demographic information and medical history, and underwent clinical and eye examinations.32 Age was defined as age at eye examination. The majority of the participants had height and weight measured at the time of the eye examination (1,502 of 2,091 persons=71.8%), and the rest of the anthropometry data were derived from previous records in the BATS (71 persons). Twins from the BATS also had additional prior extra data collected including blood pressure, total cholesterol, HDL-cholesterol, fasting blood glucose, etc, and on average 50% of these measurements were collected four years before the eye examination. Mean arterial blood pressure (MABP) was calculated as two-thirds of the diastolic blood pressure plus one third of the systolic value. Body mass index (BMI) was calculated as kg/m2.
Statistical Analysis
Both right and left eye retinal arteriolar caliber and venular caliber followed approximately normal distribution and were analysed as quantitative traits. We compared means and variance differences of covariates such as age, MABP, BMI, total cholesterol, fasting blood glucose, and retinal vascular caliber by sex among different samples.
Variance component modeling was performed using the software package Mx33 to estimate the proportion of variance explained by genetic and environmental effects. Total residual phenotypic variance of retinal arteriolar and venular caliber was partitioned into additive (A) and dominant (D) genetic variance, common (C) and unique environment (E) variance for the right and left eyes in bivariate path models. Parameter estimates for right and left eyes were tested and set to equal in all models.
Age, sex, and BMI were used as covariates in the model fitting for the TEST sample. We replaced the missing covariate data using the mean value of the covariate; therefore there was no compromise with regard to the sample size. Although those missing covariates replaced by the mean value will not contribute to the covariate effects, for this analysis conducted in a relatively young population where the genetic contribution may be stronger and environmental factors may have less influence (e.g., BATS), this impact may be minimal. Replacing of the missing values also has additional advantages such as to allowing comparison to previously published work. We included MABP, fasting glucose, cholesterol, and HDL-cholesterol as additional covariates in model fitting for the BATS. The heritability was then estimated as the proportion of genetic variance that was attributable to the total residual phenotypic variance.
In our study sample, the second set of twins, triplets and non-twin siblings in some families were treated as singletons to avoid complexity of the within-family relationship; therefore they did not contribute to the covariance in the model fitting. However, these participants have still contributed to estimation of the mean and variance and thus stabilized the estimates of twin correlations for retinal vascular caliber.
We performed variance component linkage analysis for the mean of the left and right eye retinal vascular caliber measurements using the statistical package Merlin.34 Parameter estimates were obtained by maximum likelihood methods. We also performed a gene-dropping simulation using the Merlin program. After 1000 simulations, logarithm of the odds (LOD) scores of 1.69 and 2.04 were found to be suggestive for genome-wide linkage of retinal arteriolar and venular caliber, respectively.35 Also from the simulation, significant genome-wide linkage LOD scores were 3.30 and 3.99 for retinal arteriolar and venular caliber. This is higher than the conventional significant linkage LOD score threshold of 3.0 due to pedigree size, data distribution and genotype information.
Power calculation was performed for a continuous trait according to the method as described by Purcell et al.36 Our sample had more than 90% power to detect a linked QTL accounting for 15% or higher of the trait variation in retinal vascular caliber.
Results
There were 2,091 participants from 963 families with retinal vessel measurements for both eyes. Please see http://hyper.ahajournals.org for Supplement Table S1, which displays the descriptive statistics of the phenotypes and covariates for males and females by study site. There were no significant differences in the mean and variance of retinal vascular caliber between males and females (except of the variance of right eye retinal venular caliber in TEST). There was also no significant difference of the mean and variance of retinal vascular caliber between MZ and DZ twins (except of the mean retinal venular caliber of right eye in BATS, data not shown).
There was no significant difference in overall means or variances of retinal arteriolar caliber and venular caliber between twins and their non-twin siblings for both eyes (e.g., mean retinal arteriolar caliber of right eye for twins 164.85 [95% confidence interval (CI) 164.21-165.48] and for siblings 163.49 [161.48-165.50], P = 0.21, data not shown).
Both MABP and BMI were significantly correlated with retinal arteriolar caliber (correlation coefficients −0.21 and −0.18 for right eye, −0.15 and −0.23 for left eye, and −0.20 and −0.23 for the mean of right and left eye measurements, respectively, all P <0.05) but not correlated with retinal venular caliber. However, neither arteriolar nor venular caliber were significantly correlated with height, or other covariates including sex, cholesterol, HDL-cholesterol, and fasting glucose (correlation coefficient ranged from −0.064 to 0.092, P >0.05).
In both TEST and BATS cohort, age was inversely associated with retinal arteriolar and venular caliber (all P <0.007 for CRAE and CRVE, data not shown). In BATS cohort, MABP only had a marginal effect on retinal arteriolar caliber (P = 0.02, data not shown). Other covariates including sex, BMI, total cholesterol, HDL-cholesterol, and glucose had little effect on retinal arteriolar or venular caliber.
The correlation of retinal arteriolar caliber for right and left eye within twin 1 or twin 2 (within twin cross trait) was 0.69 in the TEST sample (0.90 for the BATS). For venular caliber, the correlation between right and left eye within twin 1 or twin 2 was 0.72 in the TEST (0.88 for the BATS). The phenotypic correlation between retinal arteriolar and venular caliber within twin 1 or twin 2 was 0.54 for the combined sample (0.58 for the TEST and 0.51 for the BATS).
Heritability Analysis
The intra-pair correlations of both retinal arteriolar and venular caliber were significantly higher among MZ twins than that among DZ twins (Table 1), suggesting that a genetic effect may be involved, and subsequently bivariate general ACE and ADE models for the retinal arteriolar and venular caliber of each twin’s right and left eyes were fitted, respectively (Table 2). The most parsimonious model for retinal arteriolar caliber was the AE model, in which an additive genetic variance (A) accounted for 59.4% (95% CI 53.2% to 64.7%) of variance in arteriolar caliber of each eye for the TEST sample (56.5%, 50.1% to 61.9% for BATS). Individual environmental factors (E) explained 9.3% (4.8% to 14.9%) of the variance for each eye in this sample (11.4%, 6.5% to 17.4% for BATS). The remaining 31.3% (27.9% to 35.0%) variance represented environmental influence specific (e) to each eye (Figure 1) (32.1%, 29.1% to 35.5% for BATS), which included measurement error.
Table 1.
Intra-pair Correlation of Retinal Vascular Caliber Estimated Using Mx
| Traits | Study Sample | Zygosity | No. of Twin Pairs | Intra-pair Correlation (95% CI) |
|---|---|---|---|---|
| Retinal arteriolar caliber | TEST | MZ | 175 | 0.60 (0.53-0.66) |
| DZ | 252 | 0.34 (0.25-0.42) | ||
| BATS | MZ | 180 | 0.74 (0.68-0.78) | |
| DZ | 261 | 0.42 (0.32-0.51) | ||
| Retinal venular caliber | TEST | MZ | 175 | 0.63 (0.57-0.69) |
| DZ | 252 | 0.28 (0.19-0.37) | ||
| BATS | MZ | 180 | 0.75 (0.70-0.79) | |
| DZ | 261 | 0.35 (0.24-0.45) |
TEST: Twins Eye Study in Tasmania; BATS: Brisbane Adolescent Twin Study; MZ: monozygotic; DZ: dizygotic; CI: confidence interval.
Table 2.
ACE/ADE Model Fitting Results from Mx for Retinal Vascular Caliber
| Trait | Sample | Models | A | C | D | E | e | -2LL | df | AIC |
|---|---|---|---|---|---|---|---|---|---|---|
| Retinal arteriolar caliber | TEST | ADE | 59.4 | -- | 0.0 | 9.3 | 31.3 | 4342.5 | 1877 | 588.5 |
| ACE | 49.3 | 9.5 | -- | 10.1 | 31.1 | 4341.4 | 1877 | 587.4 | ||
| AE* | 59.4 | -- | -- | 9.3 | 31.3 | 4342.5 | 1878 | 586.5 | ||
| BATS | ADE | 56.5 | -- | 0.0 | 11.4 | 32.1 | 5659.0 | 2340 | 979.0 | |
| ACE | 56.5 | 0.0 | -- | 11.4 | 32.1 | 5658.8 | 2340 | 978.8 | ||
| AE* | 56.5 | -- | -- | 11.4 | 32.1 | 5659.0 | 2341 | 977.0 | ||
| Retinal venular caliber | TEST | ADE | 58.6 | -- | 3.2 | 10.0 | 28.2 | 4447.1 | 1877 | 693.1 |
| ACE | 61.7 | 0.0 | -- | 10.1 | 28.2 | 4447.2 | 1877 | 693.2 | ||
| AE* | 61.7 | -- | -- | 10.1 | 28.2 | 4447.2 | 1878 | 691.2 | ||
| BATS | ADE | 45.0 | -- | 19.6 | 4.3 | 31.1 | 5594.3 | 2340 | 914.3 | |
| ACE | 64.2 | 0.0 | -- | 4.9 | 30.9 | 5595.4 | 2340 | 915.4 | ||
| AE* | 64.2 | -- | -- | 4.9 | 30.9 | 5595.4 | 2341 | 913.4 |
TEST: Twins Eye Study in Tasmania; BATS: Brisbane Adolescent Twin Study;
A: additive genetics; C: common environment; D: dominant genetics; E: unique environment; e: specific component; df: degrees of freedom; AIC: Akaike’s Information Criterion
The comparisons amongst ADE, ACE and AE models were based on the final saturated model, in which some parameters were constrained to equal and those specific latent effects, such as a, c or d were dropped.
Values used in the table for A(C|D)Ee were standardised proportion of variances (%)
Best-fit model.
Figure 1.
Path Diagram illustrating parameters specification in the Bivariate AE Model: additive genetic component (A) and unique environemet component (E) and specific (e) components of variance for CRAE and CRVE of both eyes in each twin of TEST sample
The most parsimonious model for retinal venular caliber was the AE model, additive genetic variance accounted for 61.7% (55.6% to 67.0%) of variance in retinal venular caliber of each eye for the TEST sample (64.2%, 58.7% to 68.8% for BATS). Individual environmental factors explained 10.1% (5.6% to 15.6%) of the variance for each eye in this ample (4.9%, 1.0% to 9.8% for BATS). The remaining 28.2% (25.0% to 31.6%) variance represented environmental influence specific to each eye (Figure 1) (30.9%, 27.9% to 34.2% for BATS). For the BATS, the model fitting was not improved by adding additional covariates (MABP, fasting glucose, cholesterol, HDL-cholesterol), and these additional covariates were dropped from the final model (data not shown).
Sex-limitation modeling was performed using male and female parameters for the best fitting model (AEe), and showed no evidence of sex differences for variance components in retinal arteriolar caliber. However, venular caliber showed small differences in variance components between sexes; in the combined TEST and BATS samples the genetic effect in males (63.9%) was only slightly higher than females (60.4%), whereas the individual environmental factor affecting both eyes was slightly higher in females (9.3%) than males (6.4%).
Genome-wide Linkage Analysis
Please see http://hyper.ahajournals.org for Supplement Table S2, which shows the breakdown of participants who contributed linkage data. There were 836 individuals from 381 families, consisting of 511 quasi-independent sib pairs from the BATS sample for linkage analysis.
The results of the linkage analysis for retinal vascular caliber are displayed in Figure 2 and 3, respectively. No genome-wide significant logarithm of odds (LOD) score was detected for either retinal arteriolar caliber or venular caliber. Two multipoint peaks for retinal arteriolar caliber were observed on chromosomes 3p12.3 and 8p23.1, indicating suggestive (or weaker) linkage (Table 3, Figure 2). The highest multipoint peak was a LOD score of 2.24 on chromosome 8p23.1 (genome-wide P-value=7.0×10−4, following adjustment for age, sex, MABP, BMI, fasting blood glucose, total cholesterol and HDL-cholesterol). Although these covariates had minimal effect on the heritability analyses, they were used in the linkage analysis for two reasons: the first was to match findings from other studies in the literature for comparison, and the second was to include those covariates to improve the distribution of traits.
Figure 2.
Genome-wide linkage analysis for retinal arteriolar caliber. Chromosome positions are displayed on the x-axis, and the y-axis displays the strength of evidence for linkage (logarithm of the odds).
Figure 3.
Genome-wide linkage analysis for retinal venular caliber. Chromosome positions are displayed on the x-axis, and the y-axis displays the strength of evidence for linkage (logarithm of the odds).
Table 3.
Genome Scan Multipoint Linkage Results for Retinal Vacular Caliber (Mean) with Suggestive LOD Scores
| Trait | Genomic Region | Position (cM) | Closest Marker | LOD* | P-value† |
|---|---|---|---|---|---|
| Retinal arteriolar caliber | 3p12.3 | 107.43 | D3S3681 | 1.77 | 2.0×10−3 |
| 8p23.1 | 16.35 | D8S277 | 2.24 | 7.0×10−4 | |
| Retinal venular caliber | 2p14 | 91.32 | D2S1779 | 2.69 | 2.0×10−4 |
| 9q21.13 | 90.23 | D9S175 | 2.12 | 9.0×10−4 |
LOD: logarithm of odds
Residual mean retinal arteriolar and venular caliber of right and left eyes, after adjusting for age, sex, body mass index, mean arterial blood pressure, fasting glucose, cholesterol, HDL-cholesterol
Multipoint probability
We also identified two suggestive loci for retinal venular caliber on chromosomes 2p14 and 9q21.13 (Table 3, Figure 3). The largest multipoint LOD score was 2.69 on chromosome 2p14 (genome-wide P-value = 2.0×10−4, after adjustment for age, sex, MABP, and relevant covariates). We then examined for individual family contribution to the total LOD score and found one family contributed the highest LOD score of 1.72. This family contribution was reasonable given that it was likely to have occurred due to being the most informative family with four sibs and both parents’ providing genome scan (10 independent-quasi sib pairs), rather than being an outlying family.
Discussion
In the study population comprising more than 2,000 twins and their siblings, we demonstrated that retinal arteriolar and venular caliber were heritable traits with common genetic variation accounting for approximately 60% of the normal variation in the twin population. We used the bivariate path model to partition the residual phenotypic variance, taking into account the phenotypes of four eyes and the latent variables as well.37 In a subset of this population, the genome-wide linkage analysis identified several suggestive linkage signals for both retinal arteriolar and venular caliber. The highest multipoint peak for retinal arteriolar caliber was on chromosome 8p23.1 with a LOD score of 2.24 and the largest multipoint LOD score for retinal venular caliber was 2.69 on chromosome 2p14.
Our estimation of heritability of retinal arteriolar and venular caliber was similar to but lower than that reported in a previous twin study from the Danish Twin registry, which showed heritabilities of retinal arteriolar and venular calibers were 70% (95% CI, 54% to 80%) and 83% (73% to 89%), respectively.20 The small difference in heritability estimation may be a reflection of the subtle different genetic composition of the study populations and is more probably due to differences between centers in measuring retinal caliber. We also found that the covariates (MABP, fasting blood glucose, total cholesterol and HDL-cholesterol) had limited effect on the retinal vascular caliber in the heritability estimation, which is consistent with the Danish Twin Study.20 The estimation of heritability of retinal arteriolar and venular caliber was similar between the TEST and the BATS, indicating that the results from this study may be largely generalizable to other Causation twin populations. Furthermore, there were no significant differences in the mean and variance of retinal arteriolar and venular caliber between twins and their non-twin siblings, which increase the confidence to the generalizability of the finding of twin studies to the Australian general population.
We found no evidence of significant linkage regions harboring genetic variants that influence the variation in retinal vascular caliber. Although there were a number of multipoint peaks at a suggestive linkage level, it is surprising that none of them replicated the linkage regions found in the an earlier linkage analysis from the Beaver Dam Eye Study (BDES).21 Retinal arteriolar and venular calibers showed no shared or overlapped suggestive linkage region, and were linked to distinct suggestive linkage regions on different chromosomes, which support the notion that the determinants of the variation of arterioles and venules may operate at the genetic level. Interestingly, one strong linkage signal reported in the BDES was at chromosome 3q28 and only significant for the covariance-adjusted retinal arteriolar caliber. The contrasting finding between the current study and the BDES may be due to the different study population, and some of the identified linkage signals in the previous study may partly reflect the strong covariate effect (e.g., linkage peak on chromosome 3q28 for retinal arteriolar caliber).38 Clearly, it underscores the need for future linkage studies on retinal vascular caliber.
It is noteworthy that there is a substantial phenotypic correlation between retinal arteriolar and venular caliber. Although we were unable to detect putative shared loci linked to both retinal arteriolar and venular caliber in the current study, multivariate analyses for venular and arteriolar caliber using both right and left eye information to explore their genetic and environmental overlap will be the next important step. We also observed a significant correlation of retinal arteriolar caliber with MABP, which is in line with previous reports from the general populations;1,2 a bivariate analysis looking at common genes for retinal arteriolar caliber and blood pressure in the future will provide additional insights into the shared genetic and environmental factors underlying the covariation. These findings add greater applicable credence of our current study findings to the general population.
There are multiple strengths of this study. These include the population-based sample, well-defined and reproducible measures of retinal vascular caliber, and the recruitment of non-twin siblings to increase the power of the linkage study and allow for testing the representativeness of the twin sample. A number of limitations should be considered for results interpretation. The genome-wide linkage analyses were performed in a subsample of the Brisbane twins. Larger sample sets and additional evidence are required to stabilise the point estimation for QTL mapping, generating replicable and confident results. In addition, some covariate measures (e.g. blood pressure) were collected several years prior to the eye examination and relevant covariate information was partially missing because retinal vascular caliber was not the primary study parameter and its related covariates were therefore not part of the original data collection. However, the covariate effect on the genetic analysis in this study is relatively small and an important covariate (e.g., age) was available for all participants.
Perspectives
In this large twin study, we demonstrate a strong genetic effect to the variation in retinal arteriolar and venular caliber. However, amongst the small subset of genotyped individuals, we found little evidence of a strong linkage for both traits in the genome-wide scan. Larger sample sizes and additional evidence are required to stabilise the point estimation for QTL mapping and generate more confident results.
Supplementary Material
Acknowledgments
Sources of Funding
The research reported in this article was supported by Ophthalmic Research Institute of Australia (DAM, CS, JBR), Foundation for Children Australia (TYW, DAM, CS), National Health and Medical Research Council (NHMRC), Canberra Australia (Project Grant 350415 to DAM), Clifford Craig Medical Research Trust, National Institutes of Health, National Eye Institute, RO1 EY018246-01 (TLY, DAM, CJH, NGM). CS is the recipient of a NHMRC Public Health Postgraduate Scholarship. DAM is the recipient of the Pfizer Australia Senior Research Fellowship.
The genome scans were supported by the Australian NHMRC’s Program in Medical Genomics [NHMRC-219178 to NGM, GWM] and the Center for Inherited Disease Research (CIDR) at Johns Hopkins University [NGM]. CIDR is fully funded through a federal contract from the National Institutes of Health to The Johns Hopkins University, Contract Number N01-HG-65403.
Footnotes
Conflict(s) of Interest/Disclosure
None
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