Association between EGF and EGFR Gene Polymorphisms and Susceptibility to Alopecia Areata in the Korean Population.

Dear Editor:

The roles of epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) in the pathogenesis of alopecia areata (AA) are unknown. However, several reports have suggested an association between EGF signaling and AA. In mice, EGF blocked hair follicle induction by down-regulation of signaling pathways such as Wnt, Sonic hedgehog, and bone morphogenetic protein pathways1. In human hair follicle culture, EGF and EGFR showed a capacity for inhibiting hair shaft elongation and changing the morphology to catagen growth pattern by suppressing mitotic regulators including RCC2 and Stathmin 123. A previous study reported that the use of EGFR inhibitors can cause skin inflammation and exacerbation of autoimmune diseases, and that these immune-related effects of EGFR inhibitors are due to their direct effects on the expression of the major histocompatibility complex class I and/or class II molecules4.

The role of single nucleotide polymorphisms (SNPs) of EGF and EGFR on the pathogenesis of AA has not yet been studied; however, our study suggested that EGF and EGFR could be associated with the pathogenesis of AA. The study aimed to determine whether variations in EGF and EGFR contribute to risk of AA in Korean population. Therefore, we investigated the significance of EGF and EGFR gene polymorphisms in the susceptibility to AA and to understand the pathogenesis of AA.

The study included patients who had AA and healthy control subjects and visited Kyung Hee University Hospital at Gangdong. The controls were recruited after they had been determined to be mentally and physically healthy in a general health check-up program. In this study, 231 patients with AA (105 males and 126 females, average age: 28.6±13.5 years) (Table 1) and 270 healthy controls (144 males and 126 females, average age: 35.7±11.8 years) were included. Informed consent was obtained from each subject, and the study was approved by the Institutional Review Board of Kyung Hee University Hospital at Gangdong (KHNMC IRB 2008-022). Genomic DNA was isolated from peripheral blood using a genomic DNA isolation reagent kit (Core BioSystem, Seoul, Korea).

Table 1

Clinical characteristics of study groups

Characteristic	Alopecia areata	Control
Patient number	231	270
Male/female	105/126	144/126
Age (yr)	28.6±13.5 (3~70)	35.7±11.8 (11~48)
Age of onset
<30 years	162
≥30 years	69
Family history
(+)	18
(−)	213
Type
Patch	197
Totalis or universalis	34

Values are presented as number only or mean±standard deviation (range).

Five SNPs (rs11568835 [promoter], rs11568943 [exon], rs2237051 [exon], rs11569017 [exon], and rs3756261 [promoter]) for EGF and another five SNPs (rs17337023 [exon], rs1140475 [exon], rs2293347 [exon], rs1050171 [exon], and rs6965469 [promoter]) for EGFR with a heterozygosity greater than 0.3 among SNPs located in the promoter or exon (http://www.ncbi.nlm.nih.gov/SNP) were selected. All 10 selected SNPs were included using Hardy-Weinberg Equilibrium test (HWE, p>0.05). The genotypes were determined by direct sequencing. The samples were sequenced using an ABI Prism 3730XL Analyzer (PE Applied Biosystems, Foster City, CA, USA). Sequence data were analyzed using the SeqManII software (DNASTAR Inc., Madison, WI, USA).

The HWE for the two SNPs was assessed using SNPStats (http://bioinfo.iconcologia.net/index.php)5. Multiple logistic regression models with three alternative models (codominant, dominant, and recessive)6 were calculated for the odds ratio, 95% confidence interval, and corresponding p-values, with control for gender as a covariable. SNPStats, HelixTree software (Golden Helix Inc., Bozeman, MT, USA), and SNPAnalyzer (ISTECH Inc., Goyang, Korea) were used.

Baseline characteristics of patients and controls are summarized in Table 1. Among five SNPs, one SNP (rs11569017) of EGF showed significant difference between the AA group and control group (Table 2). One SNP (rs6965469) of EGFR showed a significant difference between the AA group and control group (Table 2). There were no significant differences in expression of any of the SNPs of EGF and EGFR between early-onset AA and late-onset AA (data not shown). Moreover, none of the SNPs of EGF and EGFR showed significant differences between patients with and without familial history (data not shown). There was no significant difference associated with any of the SNPs of EGF. On the contrary, one SNP (rs17337023) of EGFR showed significant differences between patchy-type AA and alopecia totalis (AT) (data not shown).

Table 2

Logistic analysis of EGF, EGFR polymorphisms in patients with alopecia areata, and in normal control subjects

Gene	SNP	Genotype	Case (EGFR, n=260; EGF, n=287)	Control (n=231)	Codominant		Dominant		Recessive
					OR (95% CI)	p-value	OR (95% CI)	p-value	OR (95% CI)	p-value
EGFR	rs17337023				NA		NA		NA
	rs1140475	C/C	230 (88.5)	203 (87.9)	0.89 (0.49~1.62)	0.15	0.98 (0.55~1.76)	0.96	NA (0.00~NA)	0.057
		C/T	30 (11.5)	25 (10.8)
		T/T	0 (0.0)	2 (0.9)
	rs2293347	A/A	26 (10.0)	27 (11.7)	0.96 (0.65~1.42)	0.87	0.99 (0.68~1.44)	0.96	1.15 (0.64~2.09)	0.63
		G/A	118 (45.4)	100 (43.3)
		G/G	116 (44.6)	102 (44.2)
	rs1050171	A/A	5 (1.9)	3 (1.3)	0.73 (0.45~1.17)	0.33	0.71 (0.45~1.13)	0.15	0.60 (0.13~2.70)	0.5
		G/A	57 (21.9)	39 (16.9)
		G/G	198 (76.2)	187 (81.0)
	rs6965469	C/C	158 (60.8)	157 (68.0)	0.77 (0.52~1.14)	0.048*	0.72 (0.49~1.07)	0.11	0.15 (0.02~1.25)	0.037*
		T/C	95 (36.5)	71 (30.7)
		T/T	6 (2.3)	1 (0.4)
EGF	rs11568835	A/A	9 (3.1)	6 (2.6)	1.20 (0.80~1.79)	0.6	1.15 (0.78~1.71)	0.47	0.74 (0.24~2.33)	0.61
		G/A	84 (29.3)	71 (30.7)
		G/G	194 (67.6)	153 (66.2)
	rs11568943	A/A	9 (3.1)	5 (2.2)	0.76 (0.50~1.24)	0.4	0.76 (0.51~1.13)	0.18	0.76 (0.24~2.42)	0.64
		G/A	86 (30.0)	58 (25.1)
		G/G	192 (66.9)	167 (72.3)
	rs2237051	A/A	130 (45.3)	105 (45.5)	0.84 (0.57~1.24)	0.1	0.97 (0.67~1.40)	0.85	1.80 (1.00~3.26)	0.05
		A/G	131 (45.6)	93 (40.3)
		G/G	26 (9.1)	32 (13.9)
	rs11569017	A/A	183 (63.8)	166 (71.9)	0.63 (0.42~0.95)	0.0008*	0.69 (0.47~1.03)	0.068	NA (0.00~NA)	0.0025*
		A/T	103 (35.9)	59 (25.5)
		T/T	0 (0.0)	5 (2.2)
	rs3756261	A/A	187 (65.2)	165 (71.4)	0.72 (0.48~1.09)	0.29	0.73 (0.49~1.09)	0.12	0.93 (0.34~2.56)	0.89
		A/G	89 (31.0)	58 (25.1)
		G/G	11 (3.8)	7 (3.0)

Values are presented as number (%) or OR (95% CI). SNP: single nucleotide polymorphism, OR: odds ratio, CI: confidence interval, NA: not applicable. *p<0.05.

The present study is, to the best of our knowledge, the first to investigate a potential influence of the EGF and EGFR polymorphisms in patients with AA. There are amount of studies to association between EGF and EGFR gene polymorphism and internal malignancies. However there are only a few studies about the relationship between EGF and EGFR polymorphisms and autoimmune disease. One study from Taiwan reported the association between EGFR and rheumatoid arthritis (RA)7. In this study, 188 patients with RA and 128 controls were enrolled. The study revealed new information on EGFR polymorphisms (rs17337023) with regard to the association between susceptibility to development of RA and polymorphisms. The rs17337023 SNP was also found to be associated with systemic lupus erythematosus (SLE)8, endometriosis, leiomyomas9, and malignant oral keratinocytes10, in previous studies. In our study, the rs17337023 SNP also showed significant differences between patchy-type AA and AT, suggesting that rs17337023 is an important SNP that is extensively involved in the phenotype of AA as well as in other autoimmune diseases.

Polymorphism of EGF may cause compensatory expression of EGF. The elevation of EGF inhibits the induction of anagen phase and decreases hair shaft elongation. The polymorphism of EGFR may cause functional deceleration of protein-like EGFR inhibitor and thus, modulate the expression of immune molecules. In this manner, the SNPs of EGF and EGFR polymorphisms may be correlated with the pathogenesis of AA.

To our knowledge, this is the first study to demonstrate that EGF and EGFR polymorphisms are involved in the pathophysiology of AA or AA phenotypes. The genotype frequency of rs11569017 in EGF and rs6965469 in EGFR was significantly increased in patients with AA compared with the corresponding frequencies in healthy controls. In addition, rs17337023 polymorphism of EGFR may contribute to the clinical type of AA (patchy type or AT). Previous studies on RA and SLE have reported no significant clinical features according to different genotypes78.

In conclusion, EGF and EGFR polymorphisms may contribute to the increased susceptibility to AA, and may be associated with the phenotype of AA in the Korean population. In particular, the rs17337023 SNP of EGFR is a notable SNP that is involved in the phenotype of AA as well as in other autoimmune diseases.