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Ethnic and Mouse Strain Differences in Central Corneal
Thickness and Association with Pigmentation Phenotype
David P. Dimasi
1
, Alex W. Hewitt
2
, Kenneth Kagame
3
, Sam Ruvama
3
, Ludovica Tindyebwa
3
, Bastien
Llamas
1,4
, Kirsty A. Kirk
1
, Paul Mitchell
5
, Kathryn P. Burdon
1
, Jamie E. Craig
1
*
1Department of Ophthalmology, Flinders University, Adelaide, South Australia, Australia, 2Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye
and Ear Hospital, Melbourne, Victoria, Australia, 3Ruharo Eye Centre, Mbarara Municipality, Mbarara, Uganda, 4School of Earth and Environmental Sciences, University of
Adelaide, Adelaide, South Australia, Australia, 5Centre for Vision Research, Department of Ophthalmology and Westmead Millennium Institute, University of Sydney,
Westmead, New South Wales, Australia
Abstract
The cornea is a transparent structure that permits the refraction of light into the eye. Evidence from a range of studies
indicates that central corneal thickness (CCT) is strongly genetically determined. Support for a genetic component comes
from data showing significant variation in CCT between different human ethnic groups. Interestingly, these studies also
appear to show that skin pigmentation may influence CCT. To validate these observations, we undertook the first analysis of
CCT in an oculocutaneous albinism (OCA) and Ugandan cohort, populations with distinct skin pigmentation phenotypes.
There was a significant difference in the mean CCT of the OCA, Ugandan and Australian-Caucasian cohorts (Ugandan:
517.3637 mm; Caucasian: 539.7632.8 mm, OCA: 563.3637.2 mm; p,0.001). A meta-analysis of 53 studies investigating the
CCT of different ethnic groups was then performed and demonstrated that darker skin pigmentation is associated with a
thinner CCT (p,0.001). To further verify these observations, we measured CCT in 13 different inbred mouse strains and
found a significant difference between the albino and pigmented strains (p= 0.008). Specific mutations within the melanin
synthesis pathway were then investigated in mice for an association with CCT. Significant differences between mutant and
wild type strains were seen with the nonagouti (p,0.001), myosin VA (p,0.001), tyrosinase (p= 0.025) and tyrosinase
related protein (p= 0.001) genes. These findings provide support for our hypothesis that pigmentation is associated with
CCT and identifies pigment-related genes as candidates for developmental determination of a non-pigmented structure.
Citation: Dimasi DP, Hewitt AW, Kagame K, Ruvama S, Tindyebwa L, et al. (2011) Ethnic and Mouse Strain Differences in Central Corneal Thickness and
Association with Pigmentation Phenotype. PLoS ONE 6(8): e22103. doi:10.1371/journal.pone.0022103
Editor: Shukti Chakravarti, Johns Hopkins University, United States of America
Received January 19, 2011; Accepted June 16, 2011; Published August 10, 2011
Copyright: ß2011 Dimasi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors acknowledge that this study and preparation of the manuscript was made possible by funding from the following agencies: the
Ophthalmic Research Institute of Australia; Pfizer Australia; Glaucoma Australia; American Health Assistance Foundation; the Australian National Health and
Medical Research Council. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors wish to declare that Pfizer Australia, who provided partial funding, was not involved in the study design, collection, analysis
and interpretation of data, writing of the paper, and/or decision to submit for publication.
* E-mail: jamie.craig@flinders.edu.au
Introduction
The cornea is a transparent, avascular tissue that covers the
anterior surface of the eye. The cornea plays a fundamental role in
refracting light into the eye, as well as acting as a protective barrier
against the extra-ocular environment and pathogens. Interest in
central corneal thickness (CCT) has become widespread in the
recent literature as its association with several ocular and non-
ocular conditions becomes recognized. Most notably, CCT has
been identified as a risk factor for the potentially blinding disease
open-angle glaucoma [1–3], but abnormal measurements have
also been shown to manifest in anirida [4,5], Ehlers-Danlos
syndrome [6–8], Marfan syndrome [9,10] and osteogenesis
imperfecta [11,12]. In order to further understand the clinical
relevance of this trait, the mechanisms underlying normal CCT
variation need to be elucidated. Within the general population,
CCT is a normally distributed quantitative trait, with a large meta-
analysis conducted by Doughty and Zaman concluding that the
mean of 230 different datasets was 536631 mm [13]. As there is
scant evidence of any environmental factors influencing CCT, it is
probable that genetic factors play an important role in the
determination of this trait. Indeed, evidence from both twin and
familial studies indicate that CCT is highly heritable [14–16] and
recent studies have identified several genes associated with normal
variation in CCT [17–21].
Further evidence of the genetic nature of CCT comes from
numerous studies which have found that CCT varies between
different ethnic populations. Comparison of measurements from a
range of ethnic groups, including people of African descent,
American Indians/Alaskan natives, Australian Aborigines, Cauca-
sians, Hispanics and several Asian populations has provided clear
evidence that ethnicity influences CCT [22]. Whilst comparing the
results of different studies is difficult, several present similar findings.
Caucasians for example, have been consistently associated with a
thicker CCT than both their African and Australian Aboriginal
counterparts. In the absence of any confirmed environmental
factors, this data implies that population specific genetic variants
could be responsible for the variation in CCT. Given the striking
differences in skin pigmentation between these groups, it also follows
that dark skin is associated with a lower CCT.
To further investigate the relationship between human skin
pigmentation and CCT, we assessed CCT in two human
PLoS ONE | www.plosone.org 1 August 2011 | Volume 6 | Issue 8 | e22103
populations that had never previously been investigated for this
trait, a group of individuals with oculocutaneous albinism (OCA)
and a cohort from Uganda. The distinct skin pigmentation
phenotypes of the OCA and Ugandan groups allowed for analysis
of the role of pigmentation on CCT. In addition, a meta-analysis
of all published human CCT data was undertaken, which included
the Ugandan and Caucasian cohorts assessed in this study. The
purpose of the meta-analysis was to systematically investigate
whether any association between CCT and skin pigmentation can
be observed following examination of the literature. In order to
validate the observations seen in human studies, we then examined
CCT in 13 different inbred mouse strains. The pigmentation
status of the mouse coat colours was varied, with seven albino, two
black, two dilute brown and two agouti strains. This permitted a
direct examination of whether the pigmentation phenotype is
correlated with CCT. The potential association between skin
pigmentation and a transparent tissue such as the cornea is an
extremely novel finding and could potentially lead to the
identification of genes involved in new developmental pathways.
Methods
Ethics statement
Ethics approval for the research conducted on human partici-
pants was obtained from the human research ethics committees of
Flinders University/Southern Adelaide Health Service, Westmead
Millennium Institute at the University of Sydney and the Mbarara
University of Science and Technology (Application number 16/
056). Ethics approvals for work conducted on the mice used in this
study were granted by the Animal Welfare Committee of Flinders
University (Application number 608/05). All ethics approvals were
granted following written submissions. This research adhered to the
tenets of the Declaration of Helsinki and informed consent was
obtained from all participants.
Measurement of CCT in human participants
CCT was measured either by a contact ultrasound pachymeter
(Pachmate DGH55, DGH-KOI, Inc. Shermans Dale, PA, USA) or
by a non-contact slit-lamp mounted Optical Low Coherence
Reflectometry (OLCR) pachymeter (Haag-Streit, Switzerland). Prior
to contact pachymetry topical anaesthetic drops (either 0.4%
oxybuprocaine or 0.5% proxymetacaine) were administered to both
eyes 1 minute before measurement. An average of twenty-five
consecutive measurements were recorded in each eye, such that each
recording had a standard deviation ,5mm. An individual’s CCT
was calculated as mean of both eye measurements. Ultrasound and
OLCR were found to have excellent correlation (data not shown). A
detailed ocular history and ocular examination was performed on all
participants and people who had anterior segment disease or
previous refractive surgery were excluded from the study.
Ugandan participants (n = 297) were recruited from the Ruharo
Eye Centre located in the Mbarara Municipality. Caucasian
subjects (n = 956) were recruited through the Blue Mountains Eye
Study (BMES). The BMES is a population-based cohort study
investigating the aetiology of common ocular diseases among
suburban residents aged 49 years or older, living in the Blue
Mountains region, west of Sydney, New South Wales, Australia.
Participants were recruited during a recent survey performed in
2004 and the full recruitment methodology has been described
previously [23]. People with OCA (n = 22) were recruited during a
National Albinism Fellowship Conference. There were 15
confirmed cases of OCA 1A and one case of OCA 1B, whilst
the clinical diagnosis was inconclusive on the remaining subjects.
All OCA patients were of Australian Caucasian descent.
All statistical analyses were conducted using SPSS v18.0.
Differences in age and sex distribution amongst the BMES,
OCA and Ugandan cohorts were assessed using the one-way
ANOVA and chi-square procedures respectively. A two-way
analysis of covariance (ANCOVA) was used to assess if significant
differences in mean CCT were evident between the BMES, OCA
and Ugandan cohorts. The two-way ANCOVA procedure allows
for control of covariates such as age and sex.
Meta-analysis
A systematic literature search was performed to identify all
published studies that investigated central corneal thickness in
human populations. The PubMed database (National Center for
Biotechnology Information; NCBI) was explored in August 2010 by
using the following keyword string: ‘central corneal thickness’. A
total of 1,956 articles were identified in the PubMed database. Only
articles in English were reviewed. Several criteria had to be met for
an article to be included in the meta-analysis. To ensure consistency
of data, measurement of CCT had to be performed by ultrasound
pachymetry, as variation in CCT measurements has been
demonstrated between different instruments [24–26]. As there is
evidence to suggest that CCT decreases slightly with age [27–30],
only studies involving adult populations (mean age or recruitment
.18 years) were included. The study group had to comprise
normal, healthy corneas and be predominately made up of normal
eyes, although cohorts that contained a proportion of glaucomatous
or ocular hypertensive eyes at normal population rates were
permitted. An implicit statement regarding the ethnicity of the
population being investigated had to be included in the article and
the study group had to be ethnically homogeneous. Reporting of all
necessary data such as cohort size, mean CCT values and variance
was also required. There was no limit applied with regards to the
date at which the article was published. The final number of articles
that qualified for inclusion in the meta-analysis was 53, which
included the addition of the Caucasian and Ugandan data from this
study. A flow chart of the study selection process, including the
number of articles excluded at each phase, can be seen in Figure 1.
Articles that met the inclusion criteria stated above are shown in
Table S1. The Caucasian and Ugandan data from this study was
also included in the meta-analysis. Each study population was
assigned to an ethnic group based on the reported ethnicity and
geographical location of the recruitment. Geographical regions
were based on definitions used by the United Nations Population
Division (http://www.un.org/esa/population/). For African and
Caucasian ethnic groups that were recruited outside of Africa and
Europe respectively, these groups were designated ‘African
Migrant’ and ‘Caucasian Migrant’. Each ethnic group was
assigned to the ‘Light Skin’ or ‘Dark Skin’ cohorts based on the
map of native skin colour distributions compiled by Biasutti in
1941 (Figure 2) [31]. The data in this map is based on the 36-tone
chromatic scale devised by Austrian anthropologist Felix von
Luschan to assess the unexposed skin of human populations. In
general, pinkish-white skin corresponds to 1–12 on the scale; white
12–14; white-light brown 15–17; light brown 18–23; brown 24–
26; dark brown 27 or above. It was decided that the ‘Light Skin’
cohort would consist of ethnic groups that had a skin tone between
1–17 on the von Luschan scale and the ‘Dark Skin’ cohort would
consist of ethnic groups who were 18 or above. Therefore, the
groups were assigned as follows: Light Skin; Caucasian European,
Caucasian Migrant, East Asian. Dark Skin; Australian Aboriginal,
African Native, African Migrant, Hispanic, South Asian, South
East Asian. Even though the American Hispanic population can
be quite heterogeneous in terms of ancestry, a large proportion are
Influence of Pigmentation on Corneal Thickness
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descendants of Central and Southern America and as such, were
designated as having a skin tone of 18 or above.
The calculation of mean CCT and standard deviation values for
each ethnic group was performed by compiling data from each
study and weighting it according to the number of participants.
This data was used in the calculation of mean CCT and standard
deviation values for the ‘Light Skin’ and ‘Dark Skin’ groups, which
was also weighted according to the number of participants.
Comparison of the mean CCT values of the ‘Light Skin’ and
‘Dark Skin’ groups was undertaken using Student’s ttest in
Microsoft Excel 2003 and statistical significance was accepted as
p,0.05. As complete data on age and sex were not available from
all studies, correction for these variables was not performed. This
meta-analysis conformed to the Preferred Reporting Items for
Systematic Reviews and Meta-Analyses (PRISMA) guidelines [32].
Measurement of CCT in the mouse
For all strains, CCT was measured in adult female mice
between 6–10 weeks of age as this minimised the potential effect
sex and age may have on measurements. Mice were weighed then
Figure 1. Flow chart detailing the selection process for articles included in the meta-analysis.
doi:10.1371/journal.pone.0022103.g001
Figure 2. Global skin colour distribution of native populations. The colours on the map are based on the 36-tone chromatic scale devised by
Austrian anthropologist Felix von Luschan to assess the unexposed skin of human populations. The higher numbers represent darker skin colour.
Original data compiled by Biasutti 1941.
doi:10.1371/journal.pone.0022103.g002
Influence of Pigmentation on Corneal Thickness
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sedated with inhaled isoflurane before being anaesthetised by
intraperitoneal injection of 75 mg/kg ketamine and 30 mg/kg
metatomidine. Pupils of both eyes were dilated using tropicamide
drops. Animals were placed on an improvised support structure
mounted on the slit lamp to enable correct height adjustment with
the OLCR (Haag-Streit, USA). This method for CCT measure-
ment in animals has been verified previously [33]. Right eye
measurements were taken unless there was an existing problem,
such as corneal scarring, in which case the left eye was measured.
In order to prevent the corneal surface from drying out, a 2 ml
drop of saline solution was administered 30 seconds prior to taking
the first measurement. Each reading consisted of 10 measurements
with the highest and lowest measurements excluded. The
instrument then presents the mean of 8 measurements with a
standard deviation. At least 2 readings with a standard deviation of
less than 2 mm taken within 3 minutes of the administration of the
saline were required for inclusion. An overall mean measure was
then calculated for each animal.
Mouse strains and statistical analysis
Inbred mouse strains for this study were obtained from the
following institutes: Adelaide University, Adelaide, Australia (AU);
Animal Resource Centre, Perth, Australia (PARC); Canberra
Hospital, Canberra, Australia (CH); Gilles Plains Animal Resource
Centre, Adelaide, Australia (GPARC); The Jackson Laboratory,
Bar Harbor, USA (JAX); Walter and Eliza Hall Institute,
Melbourne, Australia (WEHI). The following strains of mice were
included in this study (source of strain in parentheses): 129X1/SvJ
(WEHI); A/J (JAX); AKR (PARC); BALB/c (GPARC); C3H/HeJ
(JAX); CBA/CaH (AU); C57B1/KALWRIJ (PARC); C57BL/6J
(JAX); DBA/1J (JAX); DBA/2J (JAX); FVB/NJ (PARC); NOD/
Lt (CH); SJL/J (PARC). All animals were on the same diet and
housed under identical conditions, which included a room
temperature of 21uC, 50% humidity and a 12/12 hour light/
dark cycle in the Flinders Medical Centre Animal Facility. The 13
inbred strains were classified as either albino or pigmented based
on their coat colour: Albino; 129X1/SvJ, A/J, AKR, BALB/c,
FVB/NJ, NOD/Lt, SJL/J. Pigmented; C3H/HeJ, CBA/CaH,
C57B1/KALWRIJ, C57BL/6J, DBA/1J, DBA/2J. The genotype
of each strain (excluding C57B1/KALWRIJ, as no genotype
information was available) at five known mouse coat colour genes
was determined from information found on the Jackson Labora-
tories website (http://www.jax.org/). The five genes selected were
the mouse homologue of ASIP known as nonagouti (a), myosin VA
(Myo5a), Oca2 (also known as the pgene), tyrosinase (Tyr) and
tyrosinase related protein (Tyrp1). The genotype of each mouse
strain at each gene is given in Table 1. All the mutations
investigated are recessive and the observed phenotype is
dependent on the combination with other alleles. In general each
mutation results in the following pigment phenotypes; a-black
coat, A
w
- light-bellied agouti [34]; Myo5a
d
- dilution of hair
pigmentation [35]; Oca2
P
- reduction in coat and eye pigmentation
[36]; Tyr
c
-albino [37]; Tyrp1
b
- brown coat [38].
Correlation between weight, age and CCT was assessed using
the Pearson correlation coefficient. General strain differences were
tested using a Kruskal-Wallis test. The mean CCT of pigmented
animals was compared to that of albino animals using a Mann-
Whitney U test. Statistical significance was defined as p,0.05.
Results
Human study
Data from the human cohorts is shown in Table 2. Age of
participants in the BMES ranged from 60 to 95 years, from 5 to 65
years in the OCA and from 5 to 90 in the Ugandan cohorts. There
were 62 (6.5%) confirmed cases of open-angle glaucoma in the
BMES. There was a 46 mm difference in mean CCT between the
thickest (OCA) and thinnest (Ugandan) cohorts. After adjusting for
age and gender, there was a significant difference in the mean
CCT between each of the three cohorts (p,0.001) (Table 2).
Results from the meta-analysis of human CCT data are shown in
Table 3 (also see Figure 3A). In all, 53 studies were included in the
meta-analysis with data from 76 different ethnic groups. Across all
ethnic groups there was a broad range of measurements, with a
38.4 mm difference in mean CCT between the thinnest (Australian
Aboriginals) and thickest (East Asian) groups. Following segrega-
tion of the ethnic groups into two groups based on skin
pigmentation, the mean CCT measurements were as follows:
Dark Skin = 524.6633.6 mm (n = 16,472); Light Skin = 548.46
34.1 mm (n = 14,152) (p,0.001) (Figure 3B).
Mouse study
Mean CCT readings from the 13 inbred mouse strains are
displayed in Table 4 (also see Figure 4A). Across all strains there
was a broad range of measurements, with a 24.9 mm difference in
Table 2. Characteristics of human cohorts.
BMES OCA Ugandan
p
value
N 956 22 297
Mean Age (years) 73.8 35.4 40.8
,
0.001
Gender (% Female) 59.9 72.7 46.5
,
0.001
Mean CCT 6SD (mm) 539.7
632.8
563.3
637.2
517.3
637
,
0.001
The number of participants (N), mean age in years, percentage of females and
mean CCT is shown for each cohort included in the study. Values in bold are
considered significant at the p,0.05 level. BMES = Blue Mountains Eye Study,
OCA = oculocutaneous albinism, SD = standard deviation.
doi:10.1371/journal.pone.0022103.t002
Table 1. Genotype of each mouse strain for five pigment
associated genes.
Allele at coat colour loci
Strain Coat Colour
a Myo5a Oca2 Tyr Tyrp1
129X1/SvJ Albino A
w
+Oca2
P
Tyr
c
+
A/J Albino a ++Tyr
c
Tyrp1
b
AKR Albino a ++Tyr
c
+
BALB/c Albino ++ + Tyr
c
Tyrp1
b
C3H/HeJ Agouti ++ + + +
CBA/CaH Agouti ++ + + +
C57BL/6J Black a ++++
DBA/1J Dilute Brown a Myo5a
d
++Tyrp1
b
DBA/2J Dilute Brown a Myo5a
d
++Tyrp1
b
FVB/NJ Albino ++ + Tyr
c
+
NOD/Lt Albino ++ NA Tyr
c
+
SJL/J Albino ++ Oca2
P
Tyr
c
+
For each gene, the wild-type allele is designated by the ‘+’ symbol. The ‘A
w
’
allele of the agene carried by the 129X1/SvJ strain is a distinct variant that has
been classified as wild-type for this study. No genotype information was
available for the C57BL/KALWRIJ strain. NA indicates an unavailable genotype.
doi:10.1371/journal.pone.0022103.t001
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mean CCT between the thinnest (DBA/1J) and thickest (AKR)
strains. There was a significant difference between the CCT
measurements across all strains (p,0.001). There was no
correlation between weight (p= 0.297) or age (p= 0.426) of the
mice and CCT. Following segregation of the strains into two
groups based on coat pigmentation, the mean CCT measurements
were as follows: Pigmented = 78.368.8 mm (n = 53); Albi-
no = 83.5611.1 mm (n = 79) (p= 0.008) (Figure 4B). We also
analysed the data based on the genotype status of five pigment
associated mouse genes. All mice were divided into groups based
on whether they were mutant or wild type at the particular locus
and then assessed for any differences in mean CCT (Table 5).
Significant differences were seen between mice carrying the wild
type and mutant alleles of a,Myo5a,Oca2, Tyr and Tyrp. There was
no significant difference in CCT in mice carrying the wild type
and mutant alleles of Oca2.
Discussion
A healthy cornea is essential for normal vision, as it serves a
number of roles that are vital to maintaining the structure and
function of the eye. One of the primary roles of the cornea is to
refract light into the eye, a property that is primarily due to the
transparent nature of the tissue. Another important attribute of the
cornea is the CCT, a normally distributed trait that requires
assessment for several ocular disorders and is also associated with
several non-ocular conditions. There is sufficient evidence from
hereditary and genetic studies to demonstrate that normal
variation in CCT is highly genetically determined and this is
supported by the significant differences in CCT between distinct
ethnic groups. Further analysis of the data from human ethnic
groups reveals the intriguing observation that skin pigmentation
appears to be associated with CCT, with dark skin populations
such as African Americans and Australian Aborigines consistently
exhibiting thinner CCT measurements than fairer skinned
Caucasians [22]. Our research was designed to investigate this
observation by assembling data from both human and mouse
studies, including the first reported measurements of CCT in OCA
and Ugandan populations. The outcomes from this study
demonstrate that the thickness of the cornea is intimately related
to the degree of skin or coat pigmentation.
In accordance with findings from other African and dark skin
populations, the Ugandan cohort investigated in this study had a
mean CCT that was significantly lower than our Australian-
Caucasian cohort. In comparison to other ethnic groups, the
Ugandan mean CCT of 517.3 mm was considerably thin and was
the lowest measurement found in any African study performed
using ultrasound (see Table S1). Only three studies performed with
ultrasound have observed a lower CCT than that found in the
Ugandans, with two of these from Australian Aboriginal cohorts
and the other from an Indian population [39–41]. Consequently,
the meta-analysis revealed that Australian Aboriginals have the
lowest CCT measurements of any ethnic group, although the
smaller number of participants assessed in this population suggests
that further investigation is required to confirm this finding. The
ethnic group with the second lowest CCT measurements as
determined by the meta-analysis were the South Asians, which
comprised predominately of Indians, followed by African natives,
South East Asians and African migrants, indicating that the
populations with the darkest skin pigmentation also had the lowest
CCT measurements. When all the ethnic groups were segregated
into ‘Light Skin’ and ‘Dark Skin’ cohorts, the ‘Dark Skin’ group
had a significantly thinner CCT. This result was in support of the
hypothesis that darker skin pigmentation is associated with a
thinner CCT.
The data from the OCA patients, who were collectively found
to have a significantly thicker CCT than our Australian-Caucasian
population, supports our hypothesis that skin pigmentation is
associated with CCT. OCA is a group of disorders characterised
by congenital hypopigmentation of the skin, hair and eyes. Ocular
abnormalities are also a major clinical sign of OCA and include
nystagmus, foveal hypoplasia, colour vision impairment, reduced
visual acuity and misrouting of the optic nerve fibres. Four
different forms of OCA have been described, each presenting with
varying degrees of skin and hair pigmentation. The underlying
genetic defect is also unique to each form of OCA, with mutations
in TYR, P, TYRP1 and SLC45A2 associated with OCA types 1–4
respectively [42]. Despite some genetic and phenotypic variability,
all forms of OCA present with similar ocular abnormalities. Whilst
not a distinct ethnic group, measurement of CCT in the OCA
patients made for an interesting comparison. In terms of their skin
phenotype, OCA patients have little or no skin pigmentation and
thus sit at the opposite end of the spectrum when compared to
African or Australian Aboriginal groups. The thicker CCT
measurements in the OCA group are again consistent with our
pigmentation hypothesis, as they are significantly thicker than all
races of people with darker skin, including other Caucasians. This
data from the OCA cohort may also offer insights into the genetic
mechanisms that influence CCT in humans. The majority of
Table 3. Mean CCT for each ethnic group assessed in meta-analysis.
Ethnicity Number of Studies Number of Participants Mean CCT ±SD (mm)
Australian Aboriginal 2 280 513631.5
South Asian 6 8437 517.9633.2
Native African 6 1320 524.5635.6
South East Asian 4 2459 525.6632.4
African Migrant 11 1905 530.8635.8
Caucasian Migrant 16 5040 546.2634.2
Hispanic 5 2071 546.7633.7
European Caucasian 9 5588 548.6634.5
East Asian 13 3524 551.4633.5
The cumulative number of studies and total number of participants from these studies in each ethnic group is given. A mean CCT and standard deviation (SD) was
calculated for each ethnic group with each study weighted according to size.
doi:10.1371/journal.pone.0022103.t003
Influence of Pigmentation on Corneal Thickness
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Figure 3. Graphical representation of the human CCT meta-analysis results. (A) Mean CCT of each ethnic group. Colours indicate tone of
skin pigmentation according to the chart devised by Biasutti, 1941 (see Figure 1) (B) Mean CCT of the Dark Skin (524.6633.6 mm, n = 16,472) and
Light Skin (548.4634.1 mm, n =14,152) groups based on the skin colour of the ethnic groups in Figure 1A. There was a significant difference between
the groups (p,0.001).
doi:10.1371/journal.pone.0022103.g003
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patients in this study presented with OCA1, which results from
mutations in TYR [42,43]. Whilst the exact mutation is unknown
in these patients, the results do identify TYR as a candidate gene
for future research of CCT genetics.
Data showing an association between skin pigmentation and
CCT in humans is supported by observations in the mouse.
Measurements from the 13 inbred mouse strains used in this study
definitively showed that CCT is influenced by pigmentary
differences. A highly significant association was observed when
comparing measurements across all strains (p,0.001) and there
was no correlation with weight and CCT, as these were eliminated
as potential covariates. Given our ability to control for variables
such as the type of pachymeter, pachymeter operator and
environmental conditions, it is highly probable that the genetic
diversity between the strains is responsible for the differences in
CCT. This data is consistent with the findings of Lively et al, where
a significant difference in CCT was seen across 17 strains of mice
[44]. Similarly, Henriksson et al demonstrated variation in CCT
across three inbred mouse strains [45]. While comparisons can be
made within studies, differences in measurement methodology
make it difficult to compare the CCT readings across these studies
however, with Lively et al employing ultrasound pachymetry,
Henriksson et al utilising histology and our study employing
OLCR. For example, we obtained a mean CCT measurement for
BALB/c mice of 87.666.3 mm, whilst Henriksson et al found a
mean of 134.2612.9 mm for the same strain. Whilst the small size
of the mouse eye makes accurate measurement of CCT
problematic, previous research has confirmed the utility of using
OLCR for this purpose [17,33]. Further evidence that strain
differences in CCT are genetically determined comes from a
recent linkage study that identified a quantitative trait locus for
CCT on mouse chromosome 7 [46]. If the mouse data is
extrapolated to humans, then it would suggest that genetic factors
play a large role in the CCT variation seen between ethnic groups.
The genes responsible however, remain largely unknown,
although our data from the pigment analysis may offer some clues.
Upon segregating the mouse strains into either pigmented or
albino groups, the albino animals were found to have a
significantly thicker CCT. Of the seven albino strains measured,
six had greater mean CCT measurements than the C57BL/6J
mouse, which was thickest pigmented strain. This result was
consistent with the human data and supported the hypothesis that
darker skin or coat pigmentation is associated with a thinner CCT.
The use of a murine model can also alleviate many of the
problems faced in human studies and can potentially offer a more
accurate representation of the influence that pigment has on CCT.
Our experimental design allowed us to control for variables that
may influence data from human studies, including the type of
pachymeter, pachymeter operator, sex and age. The impact of
environmental factors was also limited, with the mice kept in the
same living conditions and on the same diet. The use of inbred
mice also reduced any intra-strain variation that could result from
genetic differences. If the association between CCT and coat
pigmentation is a genuine biological interaction, then it is plausible
that genes involved in pigment biosynthesis are also involved in the
development of the cornea. To date, some 368 mouse colour loci
have been identified, of which 159 are cloned genes, ensuring
there are numerous candidate genes that could be involved in
CCT determination [47].
To identify what genes may be responsible for this variability in
CCT, genotyping information was obtained for specific mutations
within five known mouse pigment genes (http://www.jax.org/).
The goal of this analysis was to ascertain if the melanin
biosynthesis pathway was potentially linked to CCT determina-
tion. The genes investigated were a,Myo5a,Oca2,Tyr and Tyrp1.
Mutations in these genes have varying effects on pigment
expression, whilst the combination of alleles is also important to
the final phenotype of the mouse. Our results indicated that the a,
Myo5a
d
,Tyr
c
and Tyrp1
b
alleles were all significantly associated with
a difference in CCT when compared to the wild type alleles. In
particular, the a,Myo5a
d
and Tyrp1
b
alleles all showed a highly
significant association with a thinner CCT. What potential role
these genes may play in the determination of CCT however, is
unclear. The function of the nonagouti protein is to act as a ligand
for the melanocortin-1 receptor and regulate the ratio of
eumelanin to pheomelanin production, whilst Myo5a is involved
in cellular motility and mutations are believed to disrupt pigment
biosynthesis through abnormal trafficking of melanosomes within
melanocytes [48]. Tyr is the rate-limiting enzyme in the
bioysnthesis of melanin and catalyses the initial step, the
conversion of tyrosine to DOPAquinone, whereas tyrp1 is known
to catalyse the formation of an intermediate in the eumelanin
synthesis pathway and to stabilise the tyrosinase protein [49–51].
None of these protein functions appear to have any obvious
involvement in corneal development, although this probably
reflects the novelty of the association between pigment and
CCT. It is also unclear as to whether these genes are acting
independently or whether there is a combined effect on overall
pigmentation levels. Other pigment-related genes in addition to
the five investigated in this study may also be contributing to the
association with CCT. Further work is needed to verify the
relationship between a,Myo5a
d
,Tyr
c
and Tyrp1
b
and CCT,
including measurement in additional mouse strains, expression
and functional analysis and potentially, examination of these genes
in human cohorts.
The concept that genes associated with pigment regulation are
also involved in the development of a transparent tissue such as the
cornea is extremely novel. Previous data from human studies has
confirmed the presence of melanocytes and their product melanin
at the corneo-scleral junction of adults, although the function of a
pigmented limbus remains unknown [52]. Similar observations
have been made in an avian model, where melanocytes are present
in the corneal limbus of day 13 embryos, indicating that expression
Table 4. Coat colour and mean CCT of each inbred mouse
strain.
Strain Coat Colour
Number of
Animals
Mean CCT
±SD (
m
m)
DBA/1J Dilute Brown 6 70.666.9
DBA/2J Dilute Brown 5 71.162.6
C57B1/KALWRIJ Black 5 73.664.6
A/J Albino 28 74.465.8
CBA/CaH Agouti 6 79.367.4
C3H/HeJ Agouti 12 79.9612.3
C57BL/6J Black 19 82.865.6
SJL/J Albino 5 86.8624.2
FVB/NJ Albino 6 87.4613.9
BALB/c Albino 24 87.666.3
NOD/Lt Albino 6 87.966.6
129X1/SvJ Albino 4 88.666.1
AKR Albino 6 95.564.6
SD = standard deviation.
doi:10.1371/journal.pone.0022103.t004
Influence of Pigmentation on Corneal Thickness
PLoS ONE | www.plosone.org 7 August 2011 | Volume 6 | Issue 8 | e22103
of pigment genes is evident in the tissue directly adjacent to the
developing cornea [53]. Mutations in TYR, P, TYRP1 and
SLC45A2 are also known to be associated with the significant
ocular abnormalities seen in OCA patients [42], while a study by
Libby et al has demonstrated that Tyr mutations can increase the
severity of anterior segment dysgenesis in the mouse [54]. These
abnormalities indicate that genes associated with pigmentation
pathways are intimately involved in the development of ocular
structures and this may include the cornea. Given that a healthy
adult cornea is transparent, regulation of its growth by pigment
associated genes would suggest that these genes may play a more
significant role in mammalian development than what is currently
Figure 4. Graphical representation of CCT measurements conducted on the inbred mouse strains. (A) Mean CCT and coat colour of the
each individual strain. There was a significant difference in mean CCT of each strain (p,0.001). The colours of the bars represent the coat
pigmentation of the animals. Error bars indicate standard deviation. (B) Mean CCT of the Pigment (78.368.8 mm, n = 53) and Albino (83.5611.1 mm,
n = 79) groups based on the coat colour of the animals in Figure 3A. Error bars indicate standard deviation. There was a significant difference between
the groups (p= 0.008).
doi:10.1371/journal.pone.0022103.g004
Influence of Pigmentation on Corneal Thickness
PLoS ONE | www.plosone.org 8 August 2011 | Volume 6 | Issue 8 | e22103
understood. Several recent genome-wide association studies have
identified COL5A1,COL8A2,AKAP13, AVGR8, FOX01 and
ZNF469 as determinants of normal CCT variation [19–21].
However, to our knowledge there is no evidence that any of these
genes are involved in the regulation of human pigmentation. This
could be due to the fact that these studies were only performed on
ethnically homogenous populations, within which variation in
pigmentation levels is limited.
The findings presented in this paper provide further evidence
that CCT is a genetically determined trait. As seen with the ethnic
variation in humans, significant strain differences in CCT are
evident in the mouse. Furthermore, the data presented on the
correlation of pigment with CCT in both the normal and mutant
strains indicates that genes involved in the pigment pathways may
also be candidate CCT genes. The benefits of characterising these
genes lie beyond the mere identification of quantitative trait loci,
as they may offer insights into other developmental pathways that
are associated with pigmentation.
Supporting Information
Table S1 Studies included in meta-analysis of human
CCT measurements.
(DOC)
Acknowledgments
The authors are grateful to the participants from the BMES, OCA and
Ugandan cohorts and their families for consenting to this study. We would
also like to acknowledge all the ophthalmologists who assisted with this
work and to Miriam Keane for her help with the statistical analysis.
Author Contributions
Conceived and designed the experiments: DD AH K.Kagame SR LT BL
PM KB JC. Performed the experiments: DD AH K.Kagame SR LT BL
K.Kirk PM KB JC. Analyzed the data: DD AH BL KB JC. Contributed
reagents/materials/analysis tools: DD AH K.Kagame SR LT BL K.Kirk
PM KB JC. Wrote the paper: DD AH KB JC. Revised article and
approved for publication: DD AH K.Kagame SR LT BL K.Kirk PM KB
JC.
References
1. Gordon MO, Beiser JA, Brandt JD, Heuer DK, Higginbotham EJ, et al. (2002)
The Ocular Hypertension Treatment Study: baseline factors that predict the
onset of primary open-angle glaucoma. Arch Ophthalmol 120: 714–720;
discussion 829–730.
2. Miglior S, Pfeiffer N, Torri V, Zeyen T, Cunha-Vaz J, et al. (2007) Predictive
factors for open-angle glaucoma among patients with ocular hypertension in the
European Glaucoma Prevention Study. Ophthalmology 114: 3–9.
3. Francis BA, Varma R, Chopra V, Lai MY, Shtir C, et al. (2008) Intraocular
pressure, central corneal thickness, and prevalence of open-angle glaucoma: the
Los Angeles Latino Eye Study. Am J Ophthalmol 146: 741–746.
4. Brandt JD, Casuso LA, Budenz DL (2004) Markedly increased central corneal
thickness: an unrecognized finding in congenital aniridia. Am J Ophthalmol 137:
348–350.
5. Whitson JT, Liang C, Godfrey DG, Petroll WM, Cavanagh HD, et al. (2005)
Central corneal thickness in patients with congenital aniridia. Eye Contact Lens
31: 221–224.
6. Cameron JA (1993) Corneal abnormalities in Ehlers-Danlos syndrome type VI.
Cornea 12: 54–59.
7. Pesudovs K (2004) Orbscan mapping in Ehlers-Danlos syndrome. J Cataract
Refract Surg 30: 1795–1798.
8. Segev F, Heon E, Cole WG, Wenstrup RJ, Young F, et al. (2006) Structural
abnormalities of the cornea and lid resulting from collagen V mutations. Invest
Ophthalmol Vis Sci 47: 565–573.
9. Sultan G, Baudouin C, Auzerie O, De Saint Jean M, Goldschild M, et al. (2002)
Cornea in Marfan disease: Orbscan and in vivo confocal microscopy analysis.
Invest Ophthalmol Vis Sci 43: 1757–1764.
10. Heur M, Costin B, Crowe S, Grimm RA, Moran R, et al. (2008) The value of
keratometry and central corneal thickness measurements in the clinical diagnosis
of Marfan syndrome. Am J Ophthalmol 145: 997–1001.
11. Pedersen U, Bramsen T (1984) Central corneal thickness in osteogenesis
imperfecta and otosclerosis. ORL J Otorhinolaryngol Relat Spec 46: 38–41.
12. Evereklioglu C, Madenci E, Bayazit YA, Yilmaz K, Balat A, et al. (2002) Central
corneal thickness is lower in osteogenesis imperfecta and negatively correlates
with the presence of blue sclera. Ophthalmic Physiol Opt 22: 511–515.
13. Doughty MJ, Zaman ML (2000) Human corneal thickness and its impact on
intraocular pressure measures: a review and meta-analysis approach. Surv
Ophthalmol 44: 367–408.
14. Toh T, Liew SH, MacKinnon JR, Hewitt AW, Poulsen JL, et al. (2005) Central
corneal thickness is highly heritable: the twin eye studies. Invest Ophthalmol Vis
Sci 46: 3718–3722.
15. Zheng Y, Ge J, Huang G, Zhang J, Liu B, et al. (2008) Heritability of central
corneal thickness in Chinese: the Guangzhou Twin Eye Study. Invest
Ophthalmol Vis Sci 49: 4303–4307.
16. Landers JA, Hewitt AW, Dimasi DP, Charlesworth JC, Straga T, et al. (2009)
Heritability of central corneal thickness in nuclear families. Invest Ophthalmol
Vis Sci 50: 4087–4090.
17. Dimasi DP, Chen JY, Hewitt AW, Klebe S, Davey R, et al. (2010) Novel
quantitative trait loci for central corneal thickness identified by candidate gene
analysis of osteogenesis imperfecta genes. Hum Genet 127: 33–44.
18. Dimasi DP, Burdon KP, Hewitt AW, Savarirayan R, Healey PR , et al. (2010)
Candidate gene study to investigate the genetic determinants of normal variation
in central corneal thickness. Mol Vis 16: 562–569.
19. Lu Y, Dimasi DP, Hysi PG, Hewitt AW, Burdon KP, et al. (2010) Common
genetic variants near the Brittle Cornea Syndrome locus ZNF469 influence the
blinding disease risk factor central corneal thickness. PLoS Genet 6: e1000947.
20. Vitart V, Bencic G, Hayward C, Herman JS, Huffman J, et al. (2010) New loci
associated with central cornea thickness include COL5A1, AKAP13 and
AVGR8. Hum Mol Genet.
21. Vithana EN, Aung T, Khor CC, Corne s BK, Tay WT, et al. (2011) Collagen-
related genes influence the glaucoma risk factor, central corneal thickness. Hum
Mol Genet 20: 649–658.
22. Dimasi DP, Burdon KP, Craig JE (2009) The genetics of central corneal
thickness. Br J Ophthalmol.
23. Mitchell P, Smith W, Attebo K, Wang JJ (1995) Prevalence of age-related
maculopathy in Australia. The Blue Mountains Eye Study. Ophthalmology 102:
1450–1460.
24. Suzuki S, Oshika T, Oki K, Sakabe I, Iwase A, et al. (2003) Corneal thickness
measurements: scanning-slit corneal topography and noncontac t specular
Table 5. Mean CCT values for each genotype of the five pigment associated genes.
Wild Type Mutant
Gene Number Mean CCT ±SD (
m
m) Number Mean CCT ±SD (
m
m)
p
value
a63 85.3610.9 64 78.269.0
,
0.001
Myo5a 116 82.8610.3 11 70.865.1
,
0.001
Oca2 112 81.069.8 9 87.6617.6 0.121
Tyr 48 78.969.0 79 83.5611.1 0.025
Tyrp1 64 84.7611.0 63 78.869.2 0.001
The number of animals and mean CCT for each genotype of the five pigment associated genes is shown in the table. Values in bold are considered significantatthe
p,0.05 level. SD = standard deviation.
doi:10.1371/journal.pone.0022103.t005
Influence of Pigmentation on Corneal Thickness
PLoS ONE | www.plosone.org 9 August 2011 | Volume 6 | Issue 8 | e22103
microscopy versus ultrasonic pachymetry. J Cataract Refract Surg 29:
1313–1318.
25. Lackner B, Schmidinger G, Pieh S, Funovics MA, Skorpik C (2005)
Repeatability and reproducibility of central corneal thickness measurement
with Pentacam, Orbscan, and ultrasound. Optom Vis Sci 82: 892–899.
26. Zhao PS, Wong TY, Wong WL, Saw SM, Aung T (2007) Comparison of central
corneal thickness measurements by visante anterior segment optical coherence
tomography with ultrasound pachymetry. Am J Ophthalmol 143: 1047–1049.
27. Nemesure B, Wu SY, Hennis A, Leske MC (2003) Corneal thickness and
intraocular pressure in the Barbados eye studies. Arch Ophthalmol 121:
240–244.
28. Hahn S, Azen S, Ying-Lai M, Varma R (2003) Central corneal thickness in
Latinos. Invest Ophthalmol Vis Sci 44: 1508–1512.
29. Aghaian E, Choe JE, Lin S, Stamper RL (2004) Central corneal thickness of
Caucasians, Chinese, Hispanics, Filipinos, African Americans, and Japanese in a
glaucoma clinic. Ophthalmology 111: 2211–2219.
30. Suzuki S, Suzuki Y, Iwase A, Araie M (2005) Corneal thickness in an
ophthalmologically normal Japanese population. Ophthalmology 112:
1327–1336.
31. Biasutti R (1941) Le Razze e i Popol i Della Terra. Torino: Unione Tipografico.
32. Moher D, Liberati A, Tetzlaff J, Altman DG (2009) Preferred reporting items for
systematic reviews and meta-analyses: the PRISMA statement. PLoS Med 6:
e1000097.
33. Schulz D, Iliev ME, Frueh BE, Gold blum D (2003) In vivo pachymetry in
normal eyes of rat s, mice and rabbits with the optic al low coherence
reflectometer. Vision Res 43: 723–728.
34. Voisey J, van Daal A (2002) Agouti: from mouse to man, from skin to fat.
Pigment Cell Res 15: 10–18.
35. Mercer JA, Seperack PK, Strobel MC, Copeland NG, Jenkins NA (1991) Novel
myosin heavy chain encoded by murine dilute coat colour locus. Nature 349:
709–713.
36. Brilliant MH (2001) The mouse p (pink-eyed dilution) and human P genes,
oculocutaneous albinism type 2 (OCA2), and melanosomal pH. Pigment Cell
Res 14: 86–93.
37. Jackson IJ, Bennett DC (1990) Identification of the albino mutation of mouse
tyrosinase by analysis of an in vitro revertant. Proc Natl Acad Sci U S A 87:
7010–7014.
38. Zdarsky E, Favor J, Jackson IJ (1990) The molecular basis of brown, an old
mouse mutation, and of an induced revertant to wild type. Genetics 126:
443–449.
39. Landers JA, Billing KJ, Mills RA, Henderson TR, Craig JE (2007) Central
corneal thickness of indigenous Australians within Central Australia.
Am J Ophthalmol 143: 360–362.
40. Durkin SR, Tan EW, Casson RJ, Selva D, Newland HS (2007) Central corneal
thickness among Aboriginal people attending eye clinics in remote South
Australia. Clin Experiment Ophthalmol 35: 728–732.
41. Nangia V, Jonas JB, Sinha A, Matin A, Kulkarni M (2010) Central corneal
thickness and its association with ocular and general parameters in Indians: the
Central India Eye and Medical Study. Ophthalmology 117: 705–710.
42. Gronskov K, Ek J, Brondum-N ielsen K (2007) Oculocutaneous albinism.
Orphanet J Rare Dis 2: 43.
43. Tomita Y, Takeda A, Okinaga S, Tagami H, Shibahara S (1989) Human
oculocutaneous albinism caused by single base insertion in the tyrosinase gene.
Biochem Biophys Res Commun 164: 990–996.
44. Lively GD, Jiang B, Hedberg-Buenz A, Chang B, Petersen GE, et al. (2009)
Genetic Dependence of Central Corneal Thickness among Inbred Strains of
Mice. Invest Ophthalmol Vis Sci.
45. Henriksson JT, McDermott AM, Bergmanson JP (2009) Dimensions and
morphology of the cornea in three strains of mice. Invest Ophthalmol Vis Sci 50:
3648–3654.
46. Lively GD, Koehn D, Hedberg-Buenz A, Wang K, Anderson MG (2010)
Quantitative trait loci associated with murine central corneal thickness. Physiol
Genomics 42: 281–286.
47. Montoliu L, Oetting WS, Bennett DC (2010) Colour Genes. European Society
for Pigment Cell Research.
48. Wu X, Bowers B, Rao K, Wei Q, Hammer JA, 3rd (1998) Visualization of
melanosome dynamics within wild-type and dilute melanocytes suggests a
paradigm for myosin V function In vivo. J Cell Biol 143: 1899–1918.
49. Kobayashi T, Urabe K, Winder A, Jimenez-Cervantes C, Imokawa G, et al.
(1994) Tyrosinase related protein 1 (TRP1) functions as a DHICA oxidase in
melanin biosynthesis. Embo J 13: 5818–5825.
50. Jimenez-Cervantes C, Martinez-Esparza M, Solano F, Lozano JA, Garcia-
Borron JC (1998) Molecular interactions within the melanogenic complex:
formation of heterodimers of tyrosinase and TRP1 from B16 mouse melanoma.
Biochem Biophys Res Commun 253: 761–767.
51. Kobayashi T, Imokawa G, Bennett DC, Hearing VJ (1998) Tyrosinase
stabilization by Tyrp1 (the brown locus protein). J Biol Chem 273:
31801–31805.
52. Higa K, Shimmura S, Miyashita H, Shimazaki J, Tsubota K (2005) Melanocytes
in the corneal limbus interact with K19-positive basal epithelial cells. Exp Eye
Res 81: 218–223.
53. Campbell S (1989) Melanogen esis of avian neural crest cells in vitro is influenced
by external cues in the periorbital mesenchyme. Development 106: 717–726.
54. Libby RT, Smith RS, Savinova OV, Zabaleta A, Martin JE, et al. (2003)
Modification of ocular defects in mouse developmental glaucoma models by
tyrosinase. Science 299: 1578–1581.
Influence of Pigmentation on Corneal Thickness
PLoS ONE | www.plosone.org 10 August 2011 | Volume 6 | Issue 8 | e22103
