© 2001 Oxford University Press
Human Molecular Genetics, 2001, Vol. 10, No. 16 1701–1708
The melanocortin-1-receptor gene is the major freckle
gene
Maarten Bastiaens, Jeanette ter Huurne, Nelleke Gruis, Wilma Bergman, Rudi Westendorp1,
Bert-Jan Vermeer and Jan-Nico Bouwes Bavinck*
Department of Dermatology and 1Department of Clinical Epidemiology, Leiden University Medical Centre, PO Box
9600, 2300 RC Leiden, The Netherlands
Received April 17, 2001; Revised and Accepted June 12, 2001
Ephelides and solar lentigines are different types of
pigmented skin lesions. Ephelides appear early in
childhood and are associated with fair skin type and
red hair. Solar lentigines appear with increasing age
and are a sign of photodamage. Both lesions are
strong risk indicators for melanoma and nonmelanoma skin cancer. Melanocortin-1-receptor
(MC1R ) gene variants are also associated with fair
skin, red hair and melanoma and non-melanoma skin
cancer. The purpose of this study was to investigate
the relationship between MC1R gene variants, ephelides and solar lentigines. In a large case-control
study, patients with melanoma and non-melanoma
skin cancer and subjects without a history of skin
cancer were studied. In all participants, the presence
of ephelides in childhood and solar lentigines by
physical examination was assessed according to
strict definitions. The entire coding sequence of the
MC1R gene was analyzed by single-strand conformation polymorphism analysis followed by sequence
analyses. Carriers of one or two MC1R gene variants
had a 3- and 11-fold increased risk of developing
ephelides, respectively (both P < 0.0001), whereas
the risk of developing severe solar lentigines was
increased 1.5- and 2-fold (P = 0.035 and P < 0.0001),
respectively. These associations were independent
of skin type and hair color, and were comparable in
patients with and without a history of skin cancer.
The population attributable risk for ephelides to
MC1R gene variants was 60%, i.e. 60% of the ephelides in the population was caused by MC1R gene
variants. A dosage effect was found between the
degree of ephelides and the number of MC1R gene
variants. As nearly all individuals with ephelides
were carriers of at least one MC1R gene variant, our
data suggest that MC1R gene variants are necessary
to develop ephelides. The results of the study also
suggest that MC1R gene variants play a role, although
less important, in the development of solar lentigines.
INTRODUCTION
Ephelides are small pigmented spots of the skin commonly
occuring in the Caucasian population, more frequently in fairskinned individuals with red or light-blond hair (1). The spots
are best visible after sun exposure and in summer. Ephelides
appear early in childhood and partly vanish with age. Fair skin,
red hair and ephelides are indicators for an increased risk of
malignant melanoma and non-melanoma skin cancer (2–4).
Ephelides must be distinguished from solar lentigines which
occur more frequently with increasing age and can be considered as a sign of photodamage (5).
The melanocortin-1-receptor (MC1R) gene plays an
important role in the genetics of human pigmentation (6–8).
Stimulation of MC1R by α-melanocyte-stimulating hormone
(α-MSH) and other pro-opiomelanocortin (POMC) peptides
lead to enhanced adenylate cyclase and cAMP resulting in
synthesis of the black photoprotective eumelanin pigment
instead of the red phaeomelanin (9). Phaeomelanin which may
contribute to skin carcinogenesis by producing free radicals in
response to ultraviolet radiation (10) is predominantly present
in individuals with red hair and fair skin, which may explain
the sun sensitivity and inability to tan of these individuals (11).
The human MC1R gene, localized on chromosome 16q24.3
(12,13), is highly polymorphic in the white population (14) and
MC1R gene variants have been found to be associated with fair
skin and red hair (15–19). Moreover, MC1R gene variants are
associated with an increased risk of cutaneous malignant
melanoma (20–22) and non-melanoma skin cancer (17,19).
Two studies have investigated the association between
MC1R variant alleles and freckling. The first study recorded
the number of freckling sites by history up to a maximum of
seven sites including the face, shoulders, back and arms (18).
The exact meaning of ‘history’, however, was not specified.
They found a significant association (P < 0.0001) between
MC1R gene variants and the number of freckling sites. The
second study recorded levels of UV-induced skin damage indicated, for example, by back freckling (17). Significant associations between any MC1R gene variants and the red hair color
variant alleles (Arg151Cys, Arg160Trp and Asp294His) were
found. Although the freckling was not specified, the first study
probably investigated ephelides and the second study solar
lentigines.
*To whom correspondence should be addressed. Tel: +31 71 5262421; Fax: +31 71 5248106; Email: [email protected]
1702 Human Molecular Genetics, 2001, Vol. 10, No. 16
The close relationship between red hair, fair skin and
ephelides also prompted us to investigate the association
between MC1R gene variants and ephelides. Because solar
lentigines must be differentiated from ephelides, we also
studied the association between MC1R gene variants and
solar lentigines and compared this association with the
former one.
RESULTS
Composition of study population
The general characteristics of the study group were described
by De Hertog et al. (23). In short, 1019 participants were interviewed and examined at the Dermatology out-patient clinic of
the Leiden University Medical Centre. Of the subjects who
attended the study, 57 were excluded because they did not
fulfill the inclusion criteria. Among these, in four individuals
MC1R genotyping was not successful because no DNA or
PCR product could be obtained. The final series for analysis
comprised 962 subjects: 124 subjects with melanoma, 161
subjects with squamous cell carcinoma, 300 with nodular
basal cell carcinoma, 151 with superficial multifocal basal
cell carcinoma and 385 participants without a history of
skin cancer. A total of 140 subjects had more than one type
of skin cancer (23).
Characteristics of subjects with ephelides and solar
lentigines
The characteristics of the subjects with ephelides in childhood,
ephelides by physical examination and solar lentigines by
physical examination are shown in Table 1. Of all 962 participants, 378 (39.3%) had ephelides in childhood. Ephelides in
childhood and by physical examination were significantly
more common among all cancer groups as compared to the
control subjects (P < 0.05), although the association between
ephelides by physical examination and skin cancer was less
strong. Of the 962 subjects, 879 (91.4%) had solar lentigines
by physical examination and 474 (49.3%) had severe solar
lentigines. Solar lentigines were significantly more common
among patients with non-melanoma skin cancers. These data
illustrate the relationship between ephelides and solar lentigines
and skin cancer.
Of all 378 participants with ephelides in childhood, 192
(50.8%) had ephelides by physical examination. Of the 519
participants who had no history of ephelides in childhood, 61
(10.6%) showed ephelides by physical examination.
Association between the most common MC1R gene
variants and ephelides and solar lentigines
Of the 27 MC1R gene variants which were found, the nine
most common were Val60Leu, Asp84Glu, Val92Met,
Arg142His, Arg151Cys, Arg160Trp, Arg163Gln, His260Pro
and Asp294His (19). Eighteen variants showed a frequency
<0.5% of total alleles. Of the total study population, 261
(27.1%) subjects had no MC1R gene variants, 440 (45.7%) had
one MC1R gene variant, 251 (26.1%) had two MC1R gene
variants and 10 (1.1%) had three MC1R gene variants. The group
with more than one variant allele comprised 37 homozygotes and
224 compound heterozygotes i.e. two different variant alleles.
Of the 1924 alleles, only 11 showed two variants within the
same allele which occurred in 11 different individuals. Ten of
these individuals also showed a variant in the other allele, and in
one individual the other allele was wild-type.
The combinations of alleles of all participants and the
frequency of these combinations in subjects with red hair,
ephelides in childhood and severe solar lentigines, respectively, are shown in Table 2. The genotype data indicate that the
MC1R gene variants Arg151Cys, Arg160Trp and Asp294His
homozygotes and heterozygotes contribute most to the prevalence of red hair and ephelides in childhood. The genotype
table also suggests that all other common variants act as recessive alleles for ephelides in childhood in various combinations
with other alleles.
The risks of ephelides in childhood and severe solar lentigines in carriers of one common MC1R gene variant (heterozygotes) and combined with another variant allele (compound
heterozygotes and homozygotes) are shown in Table 3. All
common MC1R gene variants were strongly associated with
ephelides in childhood. Asp294His in combination with
another MC1R gene variant showed the highest risk of
ephelides followed by His260Pro, Arg142His and
Arg151Cys in combination with another variant allele.
Because of small numbers, however, 95% confidence
intervals (95% CI) are wide. The MC1R gene variants were
also associated with solar lentigines, though less strongly.
Asp294His, Asp84Glu and His260Pro showed the highest
association with solar lentigines.
The population attributable risk for ephelides to MC1R
gene variants was 60%, which indicates that 60% of the
ephelides in the population was caused by MC1R gene
variants.
Associations between ephelides by physical examination
and MC1R gene variants were similar although somewhat
weaker compared to the associations between ephelides in
childhood and MC1R gene variants (data not shown).
Ephelides are strongly associated with MC1R gene
variants independent of skin type and hair color
Ephelides in childhood were highly associated with the presence of one [odds ratio (OR) 3.1, 95% CI 2.1–4.6] and two (OR
10.8, 95% CI 7.0–16.9) MC1R gene variants (Table 4). The
association was present in all skin types. The pooled OR
adjusted for skin type using the Mantel–Haenszel test was only
slightly lower than the crude OR, indicating that MC1R gene
variants are strongly associated with ephelides in childhood
independent of skin type. Similar results were also found for
the different hair colors (data not shown).
The presence of one or two MC1R gene variants was also
significantly associated with solar lentigines, although the
risks were significantly lower compared to ephelides (OR 1.6,
95% CI 1.2–2.2; OR 2.2, 95% CI 1.5–3.1, respectively). The
association was slightly lower in subjects with darker skin
types (Table 4). Stratification into subjects with different hair
colors revealed similar results (data not shown).
Analyses in the different subgroups consisting of subjects
with melanoma and non-melanoma skin cancer, and
subjects without a history of skin cancer, showed similar
results compared to the analyses in the total group (data not
shown).
Human Molecular Genetics, 2001, Vol. 10, No. 16 1703
Table 1. Characteristics of subjects with ephelides and solar lentigines
Ephelides at childhood [no. (%)]
Ephelides by physical examination [no. (%)]
Solar lentigines by physical examination [no. (%)]
Absent
Non-severe
Severe
Absent
Non-severe
Severe
Absent
Non-severe
Severe
Male
290 (50.5)
88 (43.1)
86 (46.7)
354 (50.1)
65 (39.9)
45 (48.4)
36 (43.4)
199 (49.3)
228 (48.1)
Female
284 (49.5)
116 (56.9)
98 (53.3)
352 (49.9)
98 (60.1)
48 (51.4)
47 (56.6)
205 (50.7)
246 (51.9)
Mean
61.1
57.6
56.1
61.2
54.3
56.0
54.2
58.4
61.3
Range
28.7–79.1
28.6–77.8
24.1–79.9
28.6–79.9
28.7–77.1
24.1–76.2
29.4–78.2
28.6–77.9
24.1–79.9
IV: Tan,
never burn
41 (7.1)
7 (3.4)
3 (1.6)
42 (6.0)
5 (3.1)
4 (4.3)
8 (9.6)
17 (4.2)
26 (5.5)
III: Tan,
sometimes
burn
293 (51.1)
65 (31.9)
24 (13.1)
310 (43.9)
52 (31.9)
20 (21.5)
29 (34.9)
175 (43.3)
178 (37.5)
II: Burn,
then tan
217 (37.8)
110 (53.9)
106 (57.6)
297 (42.1)
91 (55.8)
45 (48.4)
35 (41.2)
180 (44.6)
217 (45.8)
I: Burn,
never tan
23 (4.0)
22 (10.8)
51 (27.7)
57 (8.0)
15 (9.2)
24 (25.8)
11 (13.3)
32 (7.9)
53 (11.2)
Sex
Age (years)
Skin type
Hair color age
20a
Black
40 (7.0)
3 (1.5)
6 (3.3)
42 (6.0)
4 (2.5)
3 (3.3)
3 (3.7)
22 (5.5)
24 (5.1)
Brown
130 (22.8)
42 (20.6)
35 (19.1)
158 (22.4)
32 (19.7)
17 (18.5)
25 (30.5)
89 (22.1)
93 (19.1)
Dark blond
234 (41.0)
68 (33.3)
45 (24.6)
263 (37.3)
61 (37.6)
23 (25.0)
28 (34.1)
153 (38.0)
165 (34.9)
Light Blond 160 (28.0)
76 (37.2)
48 (26.2)
213 (30.3)
49 (30.3)
22 (23.9)
23 (28.0)
114 (28.4)
147 (31.1)
Red
7 (1.2)
15 (7.4)
49 (26.8)
28 (4.0)
16 (9.9)
27 (29.3)
3 (3.7)
24 (6.0)
44 (9.3)
No cancer
265 (46.2)
74 (36.3)
46 (25.0)
298 (42.2)
67 (41.1)
20 (21.5)
47 (56.6)
184 (45.5)
154 (32.5)
Squamous
cell
carcinoma
85
38
38
109
33
19
5
57
98
Nodular
basal cell
carcinoma
165
65
70
235
28
37
19
102
179
Superficial
basal cell
carcinoma
76
30
45
112
21
18
8
58
85
Malignant
melanoma
55
32
37
70
31
23
13
50
61
Skin cancer
stateb
In total, eight subjects were not able to recall the presence of ephelides in childhood, and solar lentigines were not recorded in two subjects. Distribution of
ephelides and solar lentigines in skin cancer patients compared with control subjects all P < 0.05, except for solar lentigines and melanoma, P = 0.19.
aNot assessed in two subjects.
bA total of 140 subjects had more than one type of skin cancer.
Degree of freckling is associated with presence of MC1R
gene variants
A significant association was found between the degree of
ephelides in childhood and the presence of MC1R gene
variants (P < 0.0001) (Fig. 1). This association showed a clear
dosage effect: the degree of freckling was positively related to
the number of MC1R gene variants. A significant dosage effect
was also seen between the degree of solar lentigines and MC1R
gene variants (P < 0.0001) (Fig. 1); however, the number of
subjects with higher degrees of solar lentigines and no variant
MC1R gene alleles was considerable.
DISCUSSION
Ephelides in childhood were strongly associated with the presence of MC1R gene variants independent of skin type and hair
color. Only 42 of the 378 subjects with ephelides had no MC1R
gene variants. Also, the degree of ephelides was associated
with the number of MC1R gene variants. Both ephelides and
1704 Human Molecular Genetics, 2001, Vol. 10, No. 16
Table 2. Proportion of red hair, ephelides in childhood and solar lentigines in genotyped participants
Val60Leu
Val60Leu
0-3-3/7
Asp84Glu
0-0-2/5
Asp84Glu Val92Met
Arg142His Arg151Cys Arg160Trp
Arg163Gln His260Pro Asp294His Rare variant Wild-type allele
0-0-0/0
Val92Met
1-6-7/16
0-3-2/4
0-3-7/9
Arg142His
0-0-0/0
0-0-0/0
0-1-1/1
1-1-0/1
Arg151Cys
2-15-14/16
0-2-2/2
1-10-9/17
1-1-1/1
5-5-4/5
Arg160Trp
3-13-12/20
1-3-3/4
1-13-8/17
5-6-4/7
14-20-11/21 5-8-8/13
Arg163Gln
0-3-2/6
0-1-2/2
0-8-5/14
0-1-1/2
1-5-3/8
2-7-4/9
0-0-1/1
His260Pro
0-1-0/1
0-0-0/0
0-1-0/2
0-0-0/0
0-0-0/0
2-2-2/2
0-0-0/0
0-0-0/0
Asp294His
1-4-2/4
0-0-0/0
0-0-1/1
0-0-0/0
2-3-3/3
0-0-0/0
0-0-0/0
0-0-0/0
0-0-0/0
Rare variant 0-4-6/8
1-1-0/1
0-5-2/7
0-0-0/0
6-8-6/11
2-5-3/7
0-0-5/5
0-1-1/1
1-1-1/2
0-3-3/8
Wild allele
1-7-8/16
0-23-36/78
0-2-2/3
5-17-23/46
6-48-65/111 0-11-19/43 2-7-7/13
1-4-5/11
1-5-12/24
1-32-44/93
2-42-102/261
The rows and columns are the MC1R genotypes on each chromosome. In the case of two variant alleles on one chromosome these alleles are counted separately.
The fraction in each cell is the proportion of individuals with red hair (n = 71), ephelides in childhood (n = 378) and severe solar lentigines (n = 474), respectively.
Table 3. Risk for ephelides in childhood and solar lentigines for the most common MC1R gene variantsa
Different alleles
Ephelides [OR (95% CI)]
Heterozygotes
Solar lentigines [OR (95% CI)]
Compound heterozygotes and
homozygotes
Heterozygotes
Compound heterozygotes and
homozygotes
Wild-type homozygotesb
1.0
1.0
1.0
1.0
Val60Leu
2.8 (1.6–4.9)
9.3 (5.1–17.1)
1.4 (0.85–2.3)
2.6 (1.5–4.4)
Asp84Glu
4.0 (1.3–12.7)
11.7 (3.1–47.6)
1.6 (0.51–4.7)
3.9 (1.1–15.2)
Val92Met
2.2 (1.2–4.2)
7.5 (4.2–13.5)
1.4 (0.79–2.4)
1.5 (0.88–2.5)
Arg142His
26.0 (5.1–178.4)
3.1 (0.22–8.8)
2.2 (0.60–8.2)
Arg151Cys
3.0 (1.5–6.4)
24.8 (12.2–51.2)
1.6 (0.79–3.1)
2.7 (1.5–4.6)
Arg160Trp
4.0 (2.3–6.7)
17.9 (9.7–33.4)
2.2 (1.4–3.6)
1.9 (1.2–3.1)
Arg163Gln
1.8 (0.78–4.0)
5.2 (2.5–10.8)
1.2 (0.61–2.5)
1.4 (0.71–2.8)
His260Pro
6.1 (1.7–21.6)
26.0 (2.8–602.4)
1.8 (0.53–6.3)
3.1 (0.48–25.0)
Asp294His
3.5 (0.78–14.7)
41.5 (5.1–908.5)
1.3 (0.33–5.0)
5.5 (1.0–38.8)
aEphelides
bReference
10.4 (0.72–296.3)
present (non-severe and severe) versus absent, solar lentigines severe versus non-severe (absent and non-severe).
category.
MC1R gene variants are common in the Dutch population. The
high prevalence of MC1R gene variants and ephelides and the
strong association between these variables indicate that the
MC1R gene contributes largely to the etiology of ephelides and
suggests the MC1R gene to be the major ephelides gene. The
population attributable risk for ephelides to MC1R gene variants was 60%, i.e. 60% of the ephelides in the population was
caused by MC1R gene variants. However, expression of the
gene is not complete as half of the carriers do not have ephelides. Smaller but still statistically significant associations
were also found between MC1R gene variants and solar lentigines. Thus, the MC1R gene also plays a role in the development
of solar lentigines.
MC1R gene variants are strongly associated with red hair
and a poor tanning response (15,18) and according to our study
determine the presence of ephelides. This suggests that in
humans the MC1 receptor is a key regulator of pigmentation
phenotype and sun sensitivity. Melanocytes, which are stimulated by α-MSH through the MC1R, synthesize the black
photoprotective eumelanin pigment instead of red phaeomelanin (9). Individuals with red hair and fair skin predominantly
synthesize phaeomelanin (11). In mice, MC1R gene variants
leading to loss of function of the receptor result in an overproduction of phaeomelanin and consequently yellow hair (24). In
this light, it would be interesting to know the relative proportions of eumelanine and phaeomelanine in ephelides. The
histology of ephelides is characterized by hyperpigmentation
of the epidermis, usually with a decrease in the number of
melanocytes (25); however, information of the eumelanine/
phaeomelanine ratio has been lacking until now.
Some of the MC1R gene variants, Val60Leu, Arg142His,
Arg151Cys, Arg160Trp and Asp294His, have been found to
Human Molecular Genetics, 2001, Vol. 10, No. 16 1705
in explaining the association between MC1R gene variants and
solar lentigines.
The clinical differentiation between ephelides and solar
lentigines on the basis of morphological features can be difficult. Solar lentigines may already appear in childhood (38).
Assessing ephelides in childhood by history may harbor the
risk of misclassification. This form of misclassification,
however, would have only weakened the associations found in
our study. Moreover, the results of our study show that the
approach we used for defining and assessing ephelides and
solar lentigines was useful in discriminating these two
pigmented spots.
The findings in this study once more indicate that ephelides and solar lentigines are different types of pigmented
lesions and clearly show differences in their etiology.
MC1R gene variants are a necessary factor to develop ephelides, whereas they play a less critical role in the etiology of
solar lentigines.
MATERIALS AND METHODS
Study population
Figure 1. (A) Relationship between degree of ephelides (see Materials and
Methods) and number of MC1R gene variants. (B) Relationship between
degree of solar lentigines (see Materials and Methods) and number of MC1R
gene variants.
be unable to stimulate cAMP production as strongly as the
wild-type receptor in response to α-MSH stimulation and
may therefore alter the function of the receptor (26,27). Of
these variants, Asp294His, Arg142His, Arg151Cys and
Arg160Trp revealed the highest associations with ephelides
in our study. His260Pro was also associated with ephelides.
A reduced binding affinity of this variant allele for α-MSH
was found (28). Therefore, MC1R gene variants encoding
proteins with altered receptor binding and/or signalling
properties seem to be most important in determining the risk
of ephelides.
Solar lentigines are considered a sign of photodamage,
although genetic factors also play a role. Solar lentigines were
found to be associated with MC1R gene variants in our study.
Ultraviolet light was found to induce the release of α-MSH and
other POMC peptides and to upregulate the expression of the
MC1 receptor in the epidermis (29,30). Therefore, it has been
suggested that α-MSH may play a role in regulating responses
to UV radiation. α-MSH may also play a role in proliferation
and differentiation of melanocytes and keratinocytes (31–35),
and in immune processes and inflammation (7,36). Because
solar lentigines are histologically characterized by epidermal
hyperplasia with functionally active melanocytes (37), these
proliferation and differentiation mechanisms may be important
The Leiden skin cancer study (LSS) is an extensive hospitalbased case-control study which started in 1997 and in which
environmental and genetic risk factors for different types of
skin cancer are studied in the Dutch population. The design of
the study has been described before (23). Shortly, the study
population consisted of subjects aged 30–80 years with histologically proven squamous cell carcinoma, nodular and superficial multifocal basal cell carcinoma, malignant melanoma
and controls. Participants without a history of skin cancer were
selected from the Ophthalmology out-patient clinic of the
Leiden University Medical Center. Only individuals with skin
types I–IV according to the classification of Fitzpatrick (39)
were included.
Assessment of ephelides and solar lentigines
Ephelides are considered as the combined outcome of pigmentary traits, sun sensitivity and sun exposure. They appear early
in childhood and partly vanish with age. Therefore, in studies
investigating risk factors for skin cancer, ephelides are usually
assessed by history for the childhood period (4,40,41).
Recording ephelides at adult age either by subjects’ own
assessment or at physical examination by a dermatologist may
harbor the risk of misclassification with solar lentigines (1). In
the present study, participants were asked if they had ephelides
in childhood (∼15 years of age) localized in the face, on the
arms or upper back. Ephelides were defined as multiple, small
(1–3 mm), pale-brown macular lesions with a poorly defined
margin which are more pronounced in summer. This was illustrated with use of a chart which was originally proposed for
classification of melanocytic nevi by Gallagher et al. (42), and
was thereafter named as a ‘freckling chart’ in the Geraldton
Skin Cancer Prevention Survey by Kricker et al. (43). The
participants were asked to rate the severity of their childhood
freckling for each location using a scale from 0 (no childhood
freckling) to 5 (very severe childhood freckling). The total
score was determined in terms of the sum of the three locations
(minimum 0, maximum 15). Eight subjects were not able to
1706 Human Molecular Genetics, 2001, Vol. 10, No. 16
Table 4. Risk of ephelides and solar lentigines dependent on MC1R gene variants stratified according to skin type
All
Skin types I and II
Pooled ORa
Skin types III and IV
Ephelides in childhood Absent (n = 576) Present (n = 378)
Absent (n = 241)
Present (n = 283)
Absent (n = 335)
Present (n = 95)
Wt/Wt
82 (34.0)
24 (8.5)
136 (40.6)
18 (19.0)
218 (37.9)
42 (11.1)
Wt/Var
275 (47.7)
163 (43.1)
112 (46.5)
113 (39.9)
163 (48.7)
50 (52.6)
Var/Var
83 (14.4)
173 (45.8)
47 (19.5)
146 (51.6)
36 (10.7)
27 (28.4)
OR (95% CI)
Wt/Var versus Wt/Wt 3.1 (2.1–4.6)
3.5 (2.0–6.0)
2.3 (1.3–4.3)
2.9 (1.9–4.4)
Var/Var versus Wt/Wt 10.8 (7.0–16.9)
10.6 (5.9–19.4)
5.7 (2.7–12.1)
8.4 (5.3–13.3)
Solar lentigines by
physical examination
Non-severe
(n = 486)
Severe
(n = 474)
Non-severe
(n = 257)
Severe
(n = 270)
Non-severe
(n = 229)
Severe
(n = 204)
Wt/Wt
159 (32.7)
102 (21.5)
70 (27.2)
36 (13.3)
89 (38.9)
66 (32.4)
Wt/Var
218 (44.9)
222 (46.8)
105 (40.9)
121 (44.8)
113 (49.3)
101 (49.5)
Var/Var
109 (22.4)
150 (31.7)
82 (31.9)
113 (41.9)
27 (11.8)
37 (18.1)
OR (95% CI)
Wt/Var versus Wt/Wt 1.6 (1.2–2.2)
2.2 (1.4–3.7)
1.2 (0.78–1.9)
1.6 (1.1–2.2)
Var/Var versus Wt/Wt 2.2 (1.5–3.1)
2.7 (1.6–4.5)
1.9 (0.98–3.5)
2.3 (1.6–3.4)
Wt, wild-type allele; Var, variant allele.
for skin type using Mantel–Haenszel weighted OR.
aAdjusted
recall the presence or absence of ephelides in childhood and
were not included in the analyses. Ephelides were also
assessed by physical examination by a dermatologist using a
standard protocol. Using the freckling chart, the severity of
ephelides by physical examination was rated.
Solar lentigines are mainly considered as an indicator of sun
damage although genetic background plays an additional role.
They can best be recorded by a dermatologist investigating
anatomic skin sites which are chronically exposed to the sun,
such as the face, the neck, the fore-arms and hands (4,41). We
recorded solar lentigines examining the face, the arms and
upper back. This was performed by a dermatologist using a
standard protocol. Solar lentigines were defined as round to
polycyclic, brown to black–brown, macular or slightly raised
lesions up to 1 cm in diameter with a sparse and scattered
distribution (1). Using the freckling chart, the severity of solar
lentigines was rated for each location using a scale from 0 (no
solar lentigines) to 5 (very severe lentigines). The total score
was determined in terms of the sum of the three locations
(minimum 0, maximum 15). In two subjects, information on
the presence of solar lentigines was not available and these
subjects were not included in the analyses.
Assessment of skin type and hair color
In assessing skin type, the subjects’ own assessments of their
propensity to sunburn and ability to tan were ascertained at
interview. Skin type was recorded according to the classification of Fitzpatrick (39) as follows: always burn, never tan (skin
type I), always burn, then tan (skin type II), always tan, sometimes burn (skin type III) and always tan, never burn (skin type
IV). Original or natural hair color was also ascertained by
history at 20 years of age and classified into five categories:
red, light blond, dark blond, brown or black.
Detection of MC1R gene variants
Genomic DNA was isolated from peripheral blood leukocytes
of all participants by routine methods (44). The MC1R gene
coding sequence (GenBank accession no. X65634) was amplified by PCR in the following reaction: 200 ng genomic template DNA, 60 mM Tris–HCl pH 10.0, 2.0 mM MgCl2, 15 mM
(NH4)2SO4, 100 µM each dGTP, dTTP, dATP and dCTP, 1 µl
[α-32P]dCTP (3000 Ci/mmol), 500 ng of each PCR primer, 2 U
AmpliTaq (Perkin Elmer-Cetus) and 10% DMSO in a total
volume of 100 µl. Samples were covered with mineral oil,
denatured for 4 min at 92°C, and passed through 33 cycles of
amplification, consisting of 50 s denaturation at 92°C, 50 s
primer annealing at 58°C, 2 min elongation at 72°C. The
amplifications were carried out in 0.5 ml tubes (Perkin Elmer).
The DNA sequences of the primers were: F-5′-CAACGACTCCTTCCTGCTTC-3′ and R-5′-TGCCCAGCACACTTAAAGC-3′. The resulting 1018 bp PCR fragment was digested by
2 U of either RsaI or MspI, and screened for mutations by
single-strand conformation polymorphism analysis (45) on a
6% polyacrylamide gel with 10% glycerol. The gels were run
at room temperature for 6 h at 28 W or 16 h at 20 W for MspI
and RsaI digests, respectively.
Sequence analysis
DNA samples for sequencing were obtained by PCR as
described above with M13-tailed MC1R gene primers
Human Molecular Genetics, 2001, Vol. 10, No. 16 1707
M13MC1R-F-5′-TGTAAAACGACGGCCAGTCAACGACTCCTTCCTGCTTC-3′ and M13MC1R-IR-5′-CAGGAAACAGCTATGACCATGAGTCACGATGCTGTGGTAGC-3′, resulting
in a 542 bp fragment, and the primers M13MC1R-IF-5′GACGTTGTAAACGACGGCCAGTACCTGCAGCTCCATGCTGTC-3′ and M13MC1R-R-5′-CAGGAAACAGCTATGACCATGATGCCCAGCACACTTAAAGC-3′, resulting in a
661 bp fragment. Sequence analysis was performed on an ABI377 automated DNA sequencer using Big-Dye Terminator
Cycle Sequencing kits (Perkin Elmer) according to the manufacturer’s instructions.
Statistical analyses and strategy of analyses
Ephelides in childhood and by physical examination and solar
lentigines by physical examination were firstly classified in
three categories: absent (score 0), non-severe (score 1–3) and
severe (score 4–15). In the analyses with MC1R gene variants,
ephelides were dichotomised into absent and present (nonsevere and severe together). Because of the high prevalence of
solar lentigines, in the analyses with MC1R gene variants these
spots were dichotomised into non-severe (absent and nonsevere together) and severe. In some analyses with MC1R gene
variants, only ephelides in childhood were used because this
was considered as the most accurate method of assessing these
spots. To investigate a dosage effect between the degree of
ephelides and solar lentigines and MC1R gene variants, the
total scores of ephelides in childhood and solar lentigines were
divided into eight categories: 0 (absent), 1–6 (scores 1 to 6) and
7 (score 7 or more).
χ2 analysis was used to compare the prevalence of ephelides
and solar lentigines in controls and in the different patient
groups. Exposure ORs with 95% CIs were calculated to estimate the relative risk for the presence of ephelides and the
severity of solar lentigines dependent on the MC1R gene variants. Analyses between MC1R gene variants and ephelides and
solar lentigines were firstly performed with the nine most
common MC1R gene variants, separately, and secondly with
all MC1R gene variants together.
In the analyses, skin type and hair color were considered
possible confounding factors and the association between all
MC1R gene variants and ephelides and solar lentigines were
therefore also analyzed in strata of skin type (skin types I/II
and III/IV) and hair color (red and non-red). All analyses were
performed in the total study group, and also in the patient
groups and in controls, separately. The population attributable
risk for ephelides to MC1R gene variants was calculated.
ACKNOWLEDGEMENTS
We would like to thank all patients and controls, and all members
of the LSS for voluntarily and enthusiastically participating in
this study. The work was supported by a grant from Zorg
Onderzoek Nederland (ZON).
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