Lee et al. Journal of Physiological Anthropology 2013, 32:16
http://www.jphysiolanthropol.com/content/32/1/16

ORIGINAL ARTICLE

Open Access

Association between melanopsin gene
polymorphism (I394T) and pupillary light reflex isdependent on light wavelength
Sang-il Lee1, Akiko Hida2, Sei-ichi Tsujimura3, Takeshi Morita4, Kazuo Mishima2 and Shigekazu Higuchi1*

Abstract
Background: Our aim was to determine the association between melanopsin gene polymorphism and pupillary
light reflex under diverse photic conditions, including different intensities and wavelengths.
Methods: A total of 195 visually corrected subjects volunteered for investigation of the melanopsin gene of single
nucleotide polymorphism (SNP) of rs1079610 (I394T). The genotype groups were TT (n = 126), TC (n = 55), and CC(n = 8), and 75 of the subjects, including subjects with TT (n = 34), TC (n = 33), and CC (n = 8) participated in our
experiment. Three monochromatic lights with peak wavelengths of 465 nm (blue), 536 nm (green), and 632 nm(red) were prepared, and each light was projected to the subjects with five intensities, 12, 13, 14, 14.5 and 15 log
photons/(cm2 s), for one minute. The pupil size of the left eye was measured under each light condition after a
1-minute adaptation.
Results: The pupils of the TC + CC genotypes (n = 38) were significantly smaller than those of the TT genotype(n = 31) under a blue (463 nm) light condition with 15 log photons/(cm2 s) (P < 0.05). In contrast, there were no
significant differences under green (536 nm) and red (632 nm) light conditions. Conversely, relative pupil
constrictions of the TC + CC genotypes were greater than those of the TT genotype under both blue and green
conditions with high intensities (14.5 and 15 log photons/(cm2 s)). In contrast, there were no significant differences
between genotype groups in pupil size and relative pupilloconstriction under the red light conditions.
Conclusions: Our findings suggest that the melanopsin gene polymorphism (I394T) functionally interacts withpupillary light reflex, depending on light intensity and, particularly, wavelength, and that under a light condition
fulfilling both high intensity and short wavelength, the pupillary light response of subjects with the C allele(TC + CC) is more sensitive to light than that of subjects with the TT genotype.
Keywords: Genotype, Human, Intrinsically photosensitive retinal ganglion cells, Melanopsin gene (OPN4),
Non-image-forming responses, Pupillary light reflex, Single nucleotide polymorphism, Steady-state pupil response,
Monochromatic light

BackgroundIn mammals, a small subset of retinal ganglion cells express the photopigment melanopsin, and they are intrinsically photosensitive (hence, intrinsically photosensitive
retinal ganglion cells, ipRGCs) [1-3]. Parallel studies
have been carried out to identify the functional roles ofmelanopsin or ipRGCs using transgenic mice, such as* Correspondence: higu-s@design.kyushu-u.ac.jp
1
Department of Human Science, Faculty of Design, Kyushu University,
4-9-1 Shiobaru, Minami-ku, Fukuoka 815-8540, Japan
Full list of author information is available at the end of the article

mice lacking rods or cones [4,5] and mice lacking melanopsin [6,7]. We now know that ipRGCs transmit
photic irradiance information to the suprachiasmatic nuclei, intergeniculate leaflet, and olivary pretectal nucleus
through the retinohypothalamic tract [8,9] and that theyplay important roles in non-image-forming responses,
including circadian photoentrainment [6,10], pupillary
light reflex (PLR) [11,12], and other behavioral andphysiological functions [13-15].
In human beings, there are large inter-individual phenotypic variations in the non-image-forming effects of© 2013 Lee et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.

Lee et al. Journal of Physiological Anthropology 2013, 32:16
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light [16-18]. For instance, it is known that dark- andlight-adapted pupil sizes in normal healthy subjects havelarge inter-individual differences [17,19,20]. What causes
these inter-individual variations? According to the database of the International HapMap Project, in humanbeings, there are some genetic variations in the melanopsin (OPN4) gene. Higuchi et al. [21] attempted todemonstrate the functional differences of OPN4 gene
polymorphism (I394T) by measuring steady-state pupil
response during exposure to light, and they revealed that
there is a functional connection between OPN4 gene
polymorphism and pupillary light response. However,the spectral sensitivity of melanopsin was not consideredin their study. Melanopsin is a vitamin A-based opsin,
and all of the opsin and vitamin A-based photoreceptors
have characteristic spectral sensitivities [22]. Parallel
studies have shown the peak sensitivity of melanopsin inother mammals to be around 480 nm [23,24], unlikerods (λmax 498 nm) and short, medium, and longwavelength cones (λmax 420, 534, and 563 nm) [25], andconsistent with human melanopsin λmax [11,26]. Thus,
we hypothesized, first, that functional differences of the
OPN4 gene polymorphism in PLR would be apparent at
a short-wavelength light but not at a long-wavelength
light related to M- and L-cone excitation and, second,that these differences depend on light intensity, as suggested by our previous study [21]. Hence, the aim of thisstudy was to determine whether OPN4 gene polymorphism in a young Japanese population is associated withsteady-state pupil size during exposure to light of various wavelengths and intensities.

Methods
Subjects

Prior to the experiment, 195 Japanese university students were gathered to investigate OPN4 gene polymorphism. This recruitment was totally different fromthat in our previous study [21]. All subjects had normal
color vision, as tested by the Ishihara color vision test.
Scalp hairs were used to extract genomic DNA samples,
Diffuserto genotype the single nucleotide polymorphism (SNP)of rs1079610 (I394T) located in the coding region. Genomic DNA was extracted from a hair using a DNA Extractor FM Kit (Wako Pure Chemical Industries, Ltd.,
Osaka, Japan). The genotype groups were classified as
TT, TC, or CC, and the numbers of subjects in thosegroups were 126, 55, and 8, respectively (six being undetermined). The T and C allele frequencies of I394T
were 81.2% and 18.8%, respectively. Genotype frequencyof I394T was consistent with the Hardy-Weinberg equilibrium (χ2 = 0.40, ns). Subjects gave written informed
consent for participation in the study, which was approved by the ethical committee of Kyushu University
and the ethics committee of the National Center of
Neurology and Psychiatry.
Seventy-five subjects, including subjects with TT(n = 34, 16 men and 18 women; 20.79 ± 2.0 years old),subjects with TC (n = 33, 16 men and 17 women;
21.03 ± 2.3 years old), and subjects with CC (n = 8,
one man and seven women; 21.33 ± 1.7 years old) volunteered for the study and participated in all of the experiments. Exclusion criteria included medication or drug
consumption and shift work.
Experimental conditions

A total of 225 modules of a three-in-one type (redgreen-blue, RGB) light-emitting diode (LED) on a square
panel (350 × 350 mm) were prepared for exposure of
subjects to monochromatic light. The intensity of lights
was controlled by analog voltage-width modulation. A
diffuser, located in front of the light device, was arranged
300 mm from subject’s eyes (Figure 1). To control therange of subject’s eye movement during the measurement, we marked five points forming a cross.
The peak wavelengths of each color condition were
measured by an illuminance spectroradiometer (CL-500A,
KONICA MINOLTA INC., Japan) in the vertical direction
from the height of the subject’s eyes. The peak wavelengths were 632 nm (red), 536 nm (green), and 465 nm(blue) with half-bandwidths of 30 to 45 nm. Five intensity
PWM

LED panel

R

G

B

300mm
ObserverInfrared camera

EMR-8

Experimental image

Figure 1 Set-up for the experiments. A total of 225 modules of a 3-in-1 type (RGB) LED on a square panel (350 × 350 mm) were prepared, andthe intensity of lights was controlled by analog voltage-width modulation. The diffuser, located in front of the light device, was 300 mm fromsubject’s eyes. Pupil diameter was measured in the left eye using an EMR-8 eye-tracking system with an infrared camera (Nac Image Technology,
Inc., Japan).

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conditions, including 12, 13, 14, 14.5 and 15 log photons/(cm2 s), were modulated at each color condition equally.
Table 1 shows the illuminance (lx) and irradiance(μW/cm2) levels of the respective light conditions.
Procedure

The blue, green, and red stimuli were presented in separate sessions, consisting of five intensities. Dark adaptation (20 minutes) preceded each run, and test stimuli
were presented in the dark for one minute in stages
from low-intensity to strong-intensity. Pupil diameter
was measured in the left eye using an EMR-8 eyetracking system with an infrared camera (Nac Image
Technology, Inc., Japan). After adaptation to each test
stimulus, the steady-state pupil size was measured twoor three times for 5 seconds with a sample rate of 60
Hz. Test stimuli were maintained during the measurements. The head of each subject was fastened to a chinrest during the measurements.
It is suggested that PLR is modulated by an endogenous circadian clock (24 hours) [27]. To minimize thesecircadian effects on PLR, experiments were performed
during the daytime between 10 am and 5 pm and thetime of each run was counterbalanced between OPN4
genotypes.
Data analysis

Six subjects (TT = 3, TC = 2, CC = 1) were excluded,
owing to outlier or unstable data. Hence, data from a
total of 69 subjects (TT = 31, TC + CC = 38) were used
for statistical analysis. In our comparison between genotype groups (TT, TC, and CC), we treated TC and CC
as one group, since our previous study showed that
pupillary light responses of subjects who were homozygous (TT genotype) were significantly different fromthose of subjects who were heterozygous (TC genotype)and homozygous (CC genotype) and that there was no
significant difference between TC and CC genotypes in
PLR [21]. Thus, we focused on determining what differences are caused by the C allele and verifying the reproducibility of results of our previous study.
The steady-state pupil data in all experimental conditions were analyzed using a repeated-measures three-way

analysis of variance (ANOVA) (IBM© SPSS© version 21)
with genotype (TT, TC + CC) as between-subject factor
and light conditions (color and intensity) as independent
variables. Bonferroni post-hoc comparisons were conducted when main effects or interactions were present.
Also, comparisons of pupil responses in all experimental
conditions between the two genotype groups were performed using a two-sided, independent-sample Student’s t test. A P value less than 0.05 was considered
statistically significant.

Results
OPN4 polymorphism and pupil size

Before the comparison between OPN4 polymorphisms,
it was important to confirm that the pupillary responses
in this study were driven by ipRGCs. Figure 2 shows thepupil sizes of all subjects in all of the test conditions. In
the analysis of pupil sizes of all subjects, there were main
effects in both color (F = 197.394; P < 0.001) and intensity (F = 1532.19; P < 0.001) and there was aninteraction between color and intensity (F = 118.961;
P < 0.01) (Figure 2). The pupil sizes under blue conditions were significantly smaller than those under red
conditions in all intensities. Similarly, the pupil sizes undergreen conditions were, except for 13 log photons/cm2 s,
also smaller than those under red conditions. In the comparison between blue and green conditions, the pupil sizes
in blue conditions were smaller than those in green conditions for all of the intensities except for the 12 log photons/cm2 s condition.
Next (Figure 3), the repeated-measures three-way
ANOVA (three levels of color × five levels of intensity ×
two groups of genotype) showed an interaction between
color, intensity, and genotype (F = 3.702; P < 0.05), although the main effects of genotype were not significant.
Results of the Student’s t test between the two groups ofgenotype showed that there was a significant difference
only under the blue condition of 15 log photons/(cm2 s),
and the pupil size of TC + CC genotypes was significantly smaller than that of TT genotype (P < 0.05).
There were no significant differences between genotypes
under green and red conditions.

Table 1 Illuminance (lux) and irradiance (μW/cm2) levels of each light condition
Blue

Green

Red

Intensity log photons/(cm2 s)

lux

μW/cm2lux

μW/cm2lux

μW/cm2

12

0.28

0.35

1.95

0.33

0.54

0.28

13

2.75

3.52

19.90

3.04

6.27

3.25

14

24.96

33.92

167.14

29.56

53.89

28.01

14.5

72.62

104.42525.22

97.74

158.2883.58

15208.33318.371391.48285.49505.54272.59

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7

BLUE

**

Page 4 of 7

(cm2 s) (P < 0.05). However, there were no significant differences between genotypes under the red conditions.

GREEN RED**

Pupil size (mm)

6
**
**

5

**

**

**

**

**

**

14

14.5

15

4
3

2
1
0

12

13

Light intensity log photons/(cm2 s)

Figure 2 Pupil sizes (mean + standard deviation) of all subjects
under five intensities of blue light (blue bars), green light(green bars), and red light (red bars). There were main effects ofcolor (P < 0.001) and intensity (P < 0.001), and there was aninteraction between color and intensity (P < 0.001). Pupil sizes underblue conditions were significantly smaller than those under theother color conditions. Pupil sizes under green conditions, except
under the 13-green condition, were significantly smaller than thoseunder red conditions. **P < 0.01.

OPN4 polymorphism and relative pupil constriction

The relative pupil constriction was calculated as the percentage of pupil area under 13 to 15 log photons/(cm2 s)
relative to the pupil area under 12 log photons/(cm2 s)of each color condition. In the relative pupil constriction (Figure 4), there were main effects in both color(F = 42.171; P < 0.001) and intensity (F = 2100.906;
P < 0.001) and there was an interaction between color,
intensity, and genotype (F = 3.901; P < 0.01). The
Student’s t test showed that the relative pupilloconstrictionof the TC + CC genotypes was larger than that of the TT
genotype under the blue conditions of 14.5 and 15 log photons/(cm2 s) (P < 0.05), and the same results were obtained
under the green conditions of 14.5 and 15 log photons/(a) Blue(c) Red(b) Green

6

Pupil size (mm)Discussion
The aim of this study was to determine the functional
differences of the OPN4 polymorphism (I394T) in PLR
by addressing the relationship with light intensity andparticularly light wavelength. First of all, it is important
to confirm that the pupillary responses to the test stimuli were driven by ipRGCs. Several studies have demonstrated that the spectral characteristics of human
ipRGCs have λmax = 480 nm [11,26,28-30]. It has also
been reported that melanopsin, included in ipRGCs, isthe primary photopigment in driving pupillary response
to high-irradiance light [31,32]. Consistent with thosefindings, our results showed that pupil sizes became
smaller as the wavelength of light became closer to blue
light (465 nm) and that pupil sizes decreased continuously with increasing intensity in both blue and green
conditions. In contrast, there was no notable change inpupil size under red light conditions of 13 to 15 log photons/(cm2 s), indicating that ipRGCs were not triggeredby red conditions. Hence, the experimental data obtained in the present study are considered to be a resultof the excitement of ipRGCs.
In the comparison of pupil sizes between genotypes, asignificant difference between TT and TC + CC genotypes was found solely under the blue light condition of
15 log photons/(cm2 s), and the pupil size of TC + CC
genotypes was smaller than that of the TT genotype.
Similarly, in our previous study, we found that OPN4
gene I394T differences in PLR were apparent under ahigh-illuminance light condition [21]. The results of thepresent study, obtained using a new sample population,strongly support the results of our previous study. A
notable result, however, is that a pupil size difference

6

TT TC+CC

6

TT TC+CC

5

5

5

4

4

4

3

3

2

2

2

1

1

1

3

0

*

12

13

14

14.5

15

Light intensity log photons/(cm2 s)

0

0

12

13

14

14.5

15

Light intensity log photons/(cm2 s)

TT TC+CC

12

13

14

14.5

15

Light intensity log photons/(cm2 s)

Figure 3 Comparison of pupil sizes (mean + standard deviation) between TT (n = 31; black bars) and TC + CC (n = 38; white bars)
genotypes. (a) Blue conditions, (b) green conditions, (c) red conditions. Pupil sizes of TC + CC were significantly smaller than those of TT underthe 15-blue condition, but there were no significant differences under green and red conditions. *P < 0.05.

Lee et al. Journal of Physiological Anthropology 2013, 32:16
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Relative pupil constriction (%)(a) Blue
70

TT

Page 5 of 7(c) Red(b) Green
*

TC+CC

70

TT

TC+CC

*

*
*

70

60

50

50

50

40

40

40

30

30

30

20

20

20

10

10

10

13

14

14.5

15

Light intensity log photons/(cm2 s)

0

13

14

14.5

TC+CC

13

14

60

60

0

TT

15

Light intensity log photons/ (cm2 s)

0

14.5

15

Light intensity log photons/(cm2 s)

Figure 4 Comparison of relative pupil constrictions (mean + standard deviation), based on 12 log photons/(cm2 s), between TT(n = 31; black bars) and TC + CC (n = 38; white bars) genotypes. (a) Blue conditions, (b) Green conditions, (c) Red conditions. There weresignificant differences between TT and TC + CC under both blue and green conditions, but there was no difference under red conditions.
*P < 0.05.

between the genotype groups only appeared under blue
light. This seems to be a consequence of the spectral
sensitivity of ipRGCs showing a peak at a short wavelength, as mentioned. Given that an association between
genotypes and pupil size was found under a condition
satisfying high intensity and short wavelength, ourfindings strongly suggest a causal relationship between
I394T and PLR.
Moreover, significant differences between genotypes inrelative pupillary constriction were found not only underblue light of high intensity, but also under green light ofhigh intensity. Action spectra of ipRGCs [11] and resultsof several studies [33,34] have shown that green regionsof light are effective for driving melanopsin, althoughmelanopsin excitement is greater under blue than green
light exposure. In this respect, our findings indicate thatthe melanopsin sensitivity of the TC + CC genotype waslarger than that of the TT genotype when melanopsin
was strongly stimulated.
It is notable that under the red light conditions, there
was no significant difference between genotype groups
in pupil size and pupil constriction, regardless of photic
intensity. This is consistent with our hypothesis andsupports our findings for genotype differences in PLR,
because the red lights (λpeak 632 nm) used in ourstudy were expected to be correlated with activationof M- and L- cones (λmax 534 and 563 nm) [25], notmelanopsin. In support of this, human beings lacking
the outer retina (i.e., blind but with normal melanopsin)were barely able or unable to detect long-wave light[32,35]. Hence, we predicted that there would be no
significant differences between genotypes under the red
light conditions.
Interestingly, there are geographic or ethnic differences in allele frequency of I394T. According to theInternational HapMap Project, C allele frequencies of

I394T are 34.2% in Europeans, 27.8% in Chinese, 17% inJapanese, and 14.2% in Nigerians. It is not clear whatcaused these allele frequency differences, but it is obviousthat the C allele frequency in Europeans is larger than thatin people living in lower-latitude regions. Given that
I394T genotype groups with the C allele were more sensitive to high-intensity lights than were the TT genotype
group in this study, it would be interesting to determine
whether the C allele is associated with biological adaptation in a photic environment. In addition, there is evidence to suggest ethnic differences in seasonal affective
disorder [36], which is assumed to be increased as a resultof the short photoperiod in winter [37]. Roecklein et al.
[38] showed that an SNP of the melanopsin gene (P10L)
was associated with prevalence of seasonal affective disorder. Although this study indicates a functional connection between OPN4 gene polymorphism and a non-imageforming process, there was not sufficient physiologicalevidence. In future work, the functional differences between
OPN4 gene polymorphisms, including I394T and P10L,
should be examined with other ethnic groups.
In terms of a selection of experimental photic stimuli
for exciting melanopsin, there are ongoing debatableproblems. For instance, our results showed that genotype differences in pupil size did not always appear
under high-intensity lights. There are some claims that
established photometric measures are inappropriate forquantifying effective light exposure for melanopsin [13],and a new measurement named ‘melanopic illuminance’(m-lux) has been suggested to predict the sensitivity ofmelanopsin to lights [39,40]. Measurement of melanopic
illuminance might be helpful to explain our findings inthis study or our future work more precisely.
We used steady-state pupil response in this study, butthere is an efficient method to assess ipRGC-driven
pupil photoresponses called the post-illumination

Lee et al. Journal of Physiological Anthropology 2013, 32:16
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pupillary response (PIPR) [11,41]. This is a response
after light offset, which means it is unknown whether
PIPR represents the ipRGC-driven pupillary response tocontinuous light exposure, namely a real light environment, that we focused on in this study.
We determined an association between PLR and
OPN4 genotype groups in this study, indicating that themelanopsin sensitivity could be different, depending on
the genotype of I394T. However, we still do not know
the functional differences of the OPN4 polymorphism(I394T) in other non-image-forming processing and how
much the genotype differences in PLR could influenceother irradiance responses. For example, ipRGCs alsointeract with light-induced melatonin suppression in human beings [42,43], and it has been reported that pupil
size is correlated with melatonin suppression [17]. Further, human circadian phase could be shifted by exposure to high-intensity light and short-wavelength light[44,45], suggesting involvement of ipRGCs in humansleep-wake patterns. In addition, researchers in the fieldof physiological anthropology, which concerns human environmental adaptation, have revealed the influence oflight on human physiological responses [46-49]. To validate our findings, it is necessary to determine the relationship between I394T and such physiological responses.

Conclusions
In conclusion, human melanopsin gene polymorphism
I394T functionally interacts with PLR depending on lightintensity and, particularly, wavelength. Our findings suggest that, under a light condition that strongly excites
melanopsin (high intensity and short wavelength), thepupillary light response of C allele subjects (TC + CC) is
more sensitive to light than TT subjects. Further, wefound that green light of high intensity, even though it
activates melanopsin less than blue light, is also effectivein eliciting a functional OPN4 genetic difference in PLR.
Abbreviations
ANOVA: Analysis of variance; ipRGCs: Iintrinsically photosensitive retinal
ganglion cells; LED: Light-emitting diode; PIPR: Post-illumination pupillary
response; PLR: Pupillary light reflex; RGB: Red-green-blue; SNP: Single
nucleotide polymorphism.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SL collected experimental data, performed statistical analysis and wrote themanuscript. SL and SH participated in the design of the study. SL and AH
carried out the molecular genetic analysis. KM, ST and TM revised themanuscript. SH supervised the study and helped to draft the manuscript.
All authors read and approved the final manuscript.
Acknowledgements
This study was supported in part by JSPS KAKENHI (24370102) and a part ofthis study is the result of “Understanding of Molecular and Environmental
Bases for Brain Health” carried out under the Strategic Research Program forBrain Sciences by the Ministry of Education, Culture, Sports, Science andPage 6 of 7

Technology of Japan. The funders had no role in study design, datacollection and analysis, decision to publish, or preparation of the manuscript.
We would like to thank Takeshi NISHI at Kyushu University, Kaoru INAMI at
Fukuoka Women’s University and especially Mie KATO at National Center of
Neurology and Psychiatry for technical assistance in genetic analysis.
Author details
1
Department of Human Science, Faculty of Design, Kyushu University,
4-9-1 Shiobaru, Minami-ku, Fukuoka 815-8540, Japan. 2Department of
Psychophysiology, National Institute of Mental Health, National Center of
Neurology and Psychiatry, 4-1-1 Ogawa-Higashi, Kodaira, Tokyo 187-8553,
Japan. 3Department of Information Science and Biomedical Engineering,Kagoshima University, 1-21-40 Korimoto Kagoshima, Kagoshima 890-0065,
Japan. 4Department of Living Environmental Science, Fukuoka women’s
University, 1-1-1, Kasumigaoka, Higashi-ku, Fukuoka 813-8529, Japan.
Received: 27 May 2013 Accepted: 30 September 2013
Published: 12 October 2013

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doi:10.1186/1880-6805-32-16
Cite this article as: Lee et al.: Association between melanopsin gene
polymorphism (I394T) and pupillary light reflex is dependent on lightwavelength. Journal of Physiological Anthropology 2013 32:16.

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