Association of ADAM33 Polymorphisms and Susceptibility to Psoriasis

Abstract

Psoriasis (PS) is a common hyperproliferative and chronic inflammatory disease of the skin, which involves both genetic and environmental factors. The A disintegrin and metalloproteinase 33 (ADAM33) gene, located on chromosome 20p13, has recently been identified as an asthma-susceptibility gene by positional cloning. Recently, it has been reported that ADAM33 contributed to PS risk in the French population and white North Americans. To observe the relationship between ADAM33 gene and PS, a case–control study was conducted in a Han Chinese population. Three polymorphic sites (T1, T2, and V4) were genotyped using polymerase chain reaction–restriction fragment length polymorphism analysis in 106 patients with PS and 125 healthy controls. We observed a decreased frequency in the CG genotype and GG genotype of ADAM33 rs2787094 (V4) in cases compared with controls (p = 0.045, odds ratio = 0.461, 95% confidence interval = 0.215–0.992, and p = 0.044, odds ratio = 0.447, 95% confidence interval = 0.203–0.987, respectively). Our data suggest that the ADAM33 gene may be associated with PS risk in the Chinese population.

Introduction

P soriasis (PS) is a common hyperproliferative and chronic inflammatory disease of the skin affecting 2–3% of the population (Bowcock and Krueger, 2005). The most prevalent form of the disease is chronic plaque PS, which is characterized by raised red scaling skin patches that appear predominantly on the elbows, knees, scalp, and lower back. These skin lesions are due to keratinocyte hyperproliferation, inflammatory-cell dermal infiltration, and new vessel formation (Barker, 1991; Bos and De Rie, 1999). The pathogenesis of PS is complex. Environmental factors and genetic basis are involved in triggering or exacerbating the disease (Gudjonsson and Elder, 2007). Environmental effects such as infection, drug exposure, climate conditions, or psychosocial stress are responsible for disease susceptibility (Tagami, 1997). Disease concordance rates are much higher in monozygotic twins compared with dizygotic twins (35–72% vs. 12–23%), and substantial incidence increases in family members of affected individuals (Bowcock and Krueger, 2005). These findings point to a strong genetic component in the development of PS. To explain the genetic basis of PS, a number of genome-wide linkage studies have been undertaken, and a variety of gene variants were shown to be associated with the risk of PS.

The A disintegrin and metalloproteinase 33 (ADAM33) gene is located on chromosome 20p13, and 37 single-nucleotide polymorphisms (SNPs) have been initially identified within the gene or nearby (Van Eerdewegh et al., 2002). ADAM33 is predicted to have protease activity and has a domain structure composed of a signal sequence, prodomain, metalloprotease domain, disintegrin domain, cysteine-rich domain, a transmembrane domain, and a cytoplasmic domain (Yoshinaka et al., 2002). Although its function is not well known, ADAM33 has a significant role in airway remodeling (Holgate et al., 2006). ADAM33 has been identified as an asthma-susceptibility gene by positional cloning (Van Eerdewegh et al., 2002) and thus may be controlled by common susceptibility genes with general effects on dermal inflammation and immunity because both PS and asthma are immune system diseases. Recently, the association between ADAM33 and PS has been reported. For example, some authors found that ADAM33 was a new PS-risk gene in French population (Lesueur et al., 2007; Siroux et al., 2008) and white North Americans (Li et al., 2009). No studies, to date, have examined the association between genetic polymorphisms in ADAM33 gene and PS in a Chinese population.

In this study, we tested the association between three SNPs of ADAM33 gene (T1 [rs2280091], T2 [rs2280090], and V4 [rs2787094]) and PS. Both T1 (12433 T/C, Met764Thr) and T2 (12462 C/T, Pro774Ser) are located in the coding region of exon19, and their single-nucleotide changes result in amino acid substitution. V4 is located in the 3′ untranslational region (3′UTR) of ADAM33. The 3′UTR of ADAM33 and domains downstream of the catalytic domain can regulate potential ADAM33 activity (Umland et al., 2003). Therefore, the purpose of this study was to investigate whether these SNPs are associated with PS in a Chinese population.

Subjects and Methods

Subjects

The case–control study consisted of 106 Chinese individuals with PS, and 125 healthy Chinese individuals who served as controls (CTR). The characteristics of the study population are shown in Table 1. All cases had severe and extensive chronic plaque PS, which were ascertained through clinical and histopathological findings by the dermatologist. Patients were recruited from Xingping Hospital between November 2005 and October 2006. One hundred and twenty-five healthy Han Chinese adults with no history of PS were designated as CTR. All CTR were healthy blood donors living in the same area. All individuals included in this study were unrelated, and none of the subjects had undergone a bone marrow transplant. This study was approved by the Ethics Committee of Sichuan University. All patients and CTR gave written informed consent for genetic studies.

Table 1.

Description of Study Population

Characteristics	PS (n = 106)	CTR (n = 125)
Sex: males, n (%)	54 (50.9)	67 (53.6)
Age (mean ± SD)	32.1 ± 14.8	34.1 ± 13.7

PS, psoriasis; CTR, controls; SD, standard deviation.

Genotyping

DNA was extracted from 2 mL of frozen whole blood using a DNA Extraction Kit (Bioteke Corporation, Beijing, China) according to the manufacturer's instructions. Genotyping for ADAM33 was carried out using polymerase chain reaction (PCR)–restriction fragment length polymorphism analysis. Three SNPs in ADAM33 (T1 [rs2280091], T2 [rs2280090], and V4 [rs2787094]) were investigated in this case–control study. SNP name, primer sequences, and PCR reaction conditions are listed in Table 2. PCR products were digested for 4 h with site-specific restriction enzymes (New England BioLabs, Ipswich, MA), following the manufacturer's protocol, and analyzed by 6% polyacrylamide gel electrophoresis. The restriction enzymes used and the size of digested PCR products are shown in Table 3. A DNA sample from an individual with heterozygous genotype, which was confirmed by DNA sequencing, was used as positive control, and deionized water was used as negative control. Hardy–Weinberg equilibrium test was performed among cases and CTR, and no SNP showed Hardy–Weinberg disequilibrium.

Table 2.

Primers and Polymerase Chain Reaction Programs for ADAM33 Polymerase Chain Reaction–Restriction Fragment Length Polymorphism Genotyping

SNP name	rs number	Alleles	Forward (F) and reverse (R) primers for PCR	PCR program
T1	rs2280091	A/G	(F) 5′-ACTCAAGGTGACTGGGTGCT-3′ (R) 5′-GAGGGCATGAGGCTCACTTG-3′	94°C for 4 min; 35 cycles, 94°C for 30 s, 63°C for 30 s, 72°C for 30 s; 72°C for 10 min
T2	rs2280090	A/G	(F) 5′-TTCTCAGGGTCTGGGAGAAA-3′ (R) 5′-GCCAACCTCCTGGACTCTTA-3′	94°C for 4 min; 35 cycles, 94°C for 30 s, 57°C for 30 s, 72°C for 30 s; 72°C for 10 min
V4	rs2787094	C/G	(F) 5′-ACACACAGAATGGGGGAGAG-3′ (R) 5′-CCAGAAGCAAAGGTCACACA-3′	94°C for 4 min; 35 cycles, 94°C for 30 s, 65°C for 30 s, 72°C for 30 s; 72°C for 10 min

SNP, single-nucleotide polymorphism; PCR, polymerase chain reaction.

Table 3.

Restriction Enzyme and Size of Digested Polymerase Chain Reaction Product

	T1	T2	V4
Enzymes	NcoI	HpyCH4 III	PstI
Size of digested PCR product (bp)	A: 140 + 260 G: 400	A: 198 + 112 G: 310	G: 168 + 206 C: 374

Statistical analyses

The allele and genotype frequencies were obtained by direct counting. Statistical significance was determined by chi-square test for differences of allele and genotype frequencies. The relative risk associated with rare alleles was analyzed by an odds ratio (OR) with a 95% confidence interval (CI). All of the statistical analyses were performed using SPSS 11.5 software package (Chicago, IL).

Results

The genotype frequencies of the ADAM33 gene polymorphisms between PS cases and healthy CTR are shown in Table 4. No significant deviation from Hardy–Weinberg equilibrium in the cases and CTR was observed. The CG and GG genotypes of ADAM33 rs2787094 decreased significantly in cases compared with CTR (p = 0.045, OR = 0.461, 95% CI = 0.215–0.992, and p = 0.044, OR = 0.447, 95% CI = 0.203–0.987, respectively). However, we did not find significant differences in the allele frequencies in rs2280091 A/G, rs2280090 A/G, and rs2787094 C/G between PS cases and healthy CTR (Table 5). Our data indicate that the CG and GG genotypes of ADAM33 rs2787094 may protect subjects form PS disease. When using the Shi's standardized coefficient D' to examine the linkage disequilibrium, no statistically significant evidence for linkage disequilibrium among these three SNPs between PS patients and healthy participants was observed.

Table 4.

Genotype Frequency Distribution and Association Study in ADAM33 Between Psoriasis and Controls

		Frequency, n (%)
SNP	Genotypes	PS (n = 106)	CTR (n = 125)	p-Value	Odds ratio (95% CI)
rs2280091	AA	94 (88.68)	106 (84.80)		1.000
	AG	12 (11.32)	19 (15.20)	0.389	0.712 (0.328–1.545)
rs2280090	AA	1 (0.94)	0
	AG	17 (16.04)	26 (20.80)
	AA + AG	18 (16.98)	26 (20.80)		1.000
	GG	88 (83.02)	99 (79.20)	0.461	1.284 (0.660–2.499)
rs2787094	CC	23 (21.70)	14 (11.20)		1.000
	CG	47 (44.34)	62 (49.60)	0.045	0.461 (0.215–0.992)
	GG	36 (33.96)	49 (39.20)	0.044	0.447 (0.203–0.987)

CI, confidence interval.

Table 5.

Allele Frequency Distribution and Association Study in ADAM33 Between Psoriasis and Controls

		Frequency, n (%)
SNP	Allele	PS (n = 106)	CTR (n = 125)	p-Value	Odds ratio (95% CI)
rs2280091	A	200 (94.34)	231 (92.40)
	G	12 (5.66)	19 (7.60)	0.406	0.729 (0.346–1.540)
rs2280090	A	19 (8.96)	26 (10.40)
	G	193 (91.04)	224 (89.60)	0.604	1.179 (0.633–2.196)
rs2787094	C	93 (43.87)	90 (36.00)
	G	119 (56.13)	160 (64.00)	0.085	0.720 (0.495–1.047)

Discussion

In this study, we investigated the ADAM33 gene polymorphisms and determined whether these genetic factors are related to the occurrence of PS in a Chinese population. Our results showed that the CG and GG genotypes of ADAM33 rs2787094 decreased significantly in patients with PS compared with CTR (p = 0.045, OR = 0.461, 95% CI = 0.215–0.992, and p = 0.044, OR = 0.447, 95% CI = 0.203–0.987, respectively), suggesting that the CG and GG genotypes of ADAM33 rs2787094 may protect subjects form PS disease.

ADAM33 belongs to a large family of molecules that encode a subgroup of disintegrin-containing and zinc-dependent metalloproteinase superfamily called the ADAM family (Van Wart and Birkedal-Hansen, 1990; Black and White, 1998; Primakoff and Myles, 2000; Gunn et al., 2002). The ADAM family is implicated in diverse biological processes, including cell–cell and cell–matrix interactions (White, 2003), cell migration (Martin et al., 2002; McFarlane, 2003), cell–cell adhesion (Hundhausen et al., 2003), myofibroblast proliferation, proteolysis (Shapiro and Owen, 2002), and signal transduction (Seals and Courtneidge, 2003). ADAM33 is expressed in most human tissues, including skin (Yoshinaka et al., 2002). It is believed that ADAM33 plays important roles in immunity, inflammatory response, and remodeling in the occurrence and development of PS by shedding growth factors, cytokines, and their receptors from the cell surface. The function of the ADAM33 gene remains unknown, but its biologic function may support that ADAM33 is related to PS.

In a previous study, Holgate et al. (2003) investigated the relationship between ADAM33 polymorphisms and asthma and found that 3′UTR polymorphism of ADAM33 was significantly associated with asthma. Both asthma and PS are immune system diseases, and autoimmune diseases share a limited number of loci (Becker et al., 1998). Therefore, we hypothesized that PS may be controlled by similar susceptibility genes, such as ADAM33. Our study confirmed the hypothesis and found that the ADAM33 V4, which was also located in the 3′UTR of ADAM33, was associated with PS risk. Our data are in agreement with the results reported by Lesueur et al. (2007), who found that ADAM33 was a susceptibility gene of PS in French families. A replicated study reported that the ADAM33 rs512625 was associated with PS in a French population. Additionally, Siroux et al. (2008) reported that rs628977 was specifically associated with early-onset PS. Li et al. (2009) reported that the ADAM33 rs597980 is associated with PS in white North Americans.

Epidemiological studies have reported the association between polymorphisms of ADAM33 and a variety of autoimmune diseases, and majority of them have focused on the T1 (rs2280091) and T2 (rs2280090). For example, some researchers reported that the ADAM33 T1 and T2 polymorphisms were associated with asthma or its partial phenotypes in Hispanic population (De Sanctis et al., 1995) and U.S. population (Puddicombe et al., 2003). Howard et al. (2003) reported that T1 was associated with asthma risk in a Korean population. However, we failed to find any association between ADAM33 T1 and T2 polymorphisms and PS in a Chinese population.

Our study has 83%, 86%, and 81% power (for rs2280091, rs2280090, and rs2787094, respectively) to detect an effect with an odds ratio of 2.3 in the PS patients and healthy CTR at the level of 0.05 with two-sided test.

This study has some limitations. First, sample size is small, which may result in false-negative results. Second, the association between ADAM33 polymorphism and environmental factors is not available in this study. Third, the significant association between SNP (V4) and PS was not observed when comparing the distribution with a 2 df chi-square test (chi = 4.71, with p = 0.09), indicating that the association needs to be confirmed. Therefore, further studies with larger samples in diverse ethnic populations are of great value.

In conclusion, we investigated the association between ADAM33 polymorphisms and the risk of PS and found that the CG and GG genotypes of ADAM33 rs2787094 decreased significantly in patients with PS compared with CTR in a Chinese population.

Footnotes

Acknowledgment

This study was supported by the Applied Basic Research Programs of Science and Technology Commission Foundation of Sichuan Province (no. 2009SZ0163).

Disclosure Statement

No competing financial interests exist.

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