doi: 10.1111/j.1744-313X.2011.01045.x Association analysis of BANK1 gene with psoriasis in Southern Han Chinese X. Zhang*,1, Z. Fei†,1, J. Wan‡, J. Xu§, B. Yu¶ & M. Guan*,§,** 95% CI: 1.05–1.81, P = 0.0203). Overall, our result Summary indicates that polymorphism in BANK1 is associated Psoriasis is a chronic inflammatory skin disease with an with susceptibility to psoriasis in Southern Han Chi- immunogenetic background. This study aimed to deter- nese. mine the association between three functional SNPs of BANK1 (rs10516487, rs17266594 and rs3733197) with psoriasis in Southern Han Chinese population by Introduction determining their frequency in 242 patients with pso- Psoriasis is a common, chronic and inflammatory skin riasis and 317 healthy individuals. The genotype disease, with different kinds of immunological disor- frequencies of the detected polymorphisms were anal- ders. The typical character is described as discrete ery- ysed in relation to the susceptibility of psoriasis. Our thematous plaques covered by a silvery white scale in data show that there is no significant difference in characteristic locations (Navarini & Trueb, 2010). genotype distribution for the three BANK1 SNPs Global incidence of psoriasis is 0.6–4.8%, and in between patients and healthy controls. The AA fre- China, it is 1.23&, possibly due to differences in quency of rs3733197 is significantly higher in patients genetic and environmental factors. Although the path- with psoriasis onset before the age of 23 than in those ogenesis of psoriasis has been attributed to the activa- with late disease onset (P = 0.0069). In addition, tion of dendritic cells, T cells and keratinocytes in the analysis on BANK1 haplotype also suggests a protective psoriatic plaque (Monteleone et al., 2010), the au- role for TGC and CAT haplotype from psoriasis (OR toantibodies are recently found to be present in psori- 0.55, 95% CI: 0.34–0.89; P = 0.0144; OR 0.62, 95% asis and psoriasis arthritis (Tagami et al., 1983; CI: 0.42–0.92; P = 0.0175), whereas CGT haplotype is Leitch & Haslock, 1997; Singh & Singh, 2010), sug- associated with increased risk of the disease (OR 1.38, gesting an effective role of B cells and probably their secreted signalling molecules in this disease progres- sion. B-cell scaffold protein with ankyrin repeats 1 * Central Laboratory, Huashan Hospital, Shanghai Medical College, (BANK 1) is a B-cell-specific scaffold protein and Fudan University, Shanghai, China, † Department of Traditional Chi- Lyn tyrosine kinase substrate that facilitates the phos- nese medicine, Huashan Hospital, Shanghai Medical College, Fudan phorylation and activation of IP3R by Lyn and sub- University, Shanghai, China, ‡ Shenzhen Key Lab for Translational sequent release of Ca2+ from endoplasmic reticulum Medicine of Dermatology, Shenzhen-PKU-HKUST Medical Center, (Kozyrev et al., 2008). Nonsynonymous SNP Shenzhen, China, § Department of Dermatology, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, China, rs10516487 (R61H), branch point-site SNP – Department of Dermatology, Shenzhen Hospital Peking University, rs17266594 and a third variant rs3733197 (A383T) Shenzhen, China, ** Department of Laboratory Medicine, Huashan in the ankyrin domain of BANK1 are functional dis- Hospital, Shanghai Medical College, Fudan University, Shanghai, ease-associated variants that contribute to the suscep- China tibility of several autoimmunity diseases, including Received 27 April 2011; revised 16 August 2011; accepted 15 Sep- SLE (Kozyrev et al., 2008; Chang et al., 2009), RA tember 2011 (Orozco et al., 2009) and systemic sclerosis (Rueda Correspondence: Ming Guan, Department of Laboratory Medicine, et al., 2010). Given important roles of B cells in Huashan Hospital, Shanghai Medical College, Fudan University, 12 Central Urumqi Road, Shanghai, China. Tel: 0086 21 52888048; autoimmune diseases and recent discovery of autoan- Fax: 0086 21 62481061; E-mail: [email protected] tibodies in psoriasis, it would be of interest to inves- Bo Yu, Department of Dermatology, Shenzhen Hospital Peking tigate the link between BANK1 polymorphisms and University, No. 1120, Lian-Hua Road, Fu-Tian District, Shen-Zhen, psoriasis in the genetic homogeneous population of Guangdong, China. Tel: 0086 755 83923333; ext 849; Han Chinese living in Guangdong Province, aiming E-mail: [email protected] to define whether this gene plays a key role in the 1 X. Zhang and Z. Fei equally contributed to this study. autoimmune disease. ª 2011 Blackwell Publishing Ltd, International Journal of Immunogenetics 38, 507–512 507 508 X. Zhang et al. Materials and methods PCR to reach plateau. The reagent contained the fol- lowing: 0.5 U Taq HS (Takara, Shiga, Japan), 2 lL 2+ 10 · PCR Buffer (Mg Free, Takara), 1.5 mM MgCl Patients 2 (Takara), 0.2 mM dNTP mixture (Takara), 0.05 lM The study involved 242 patients with psoriasis and forward primer, 0.5 lM reverse primer, 0.5 lM unla- 317 healthy controls. Patients with psoriasis (158 belled probe, 0.6 lL1· SYTO 9 dye (Invitrogen, men ⁄ 84 women, mean age: 34.43 ± 12.88 years) are Carlsbad, CA, USA) and 40 ng DNA were added to of Han Chinese ethnics living in Guangdong Province. the mix solution and then complemented by water to All patients were diagnosed to have psoriasis by at 20 lL. least two independent dermatologists in the dermatol- The PCR conditions were as follows: initial denatur- ogy department in Shenzhen Hospital Peking Univer- ation at 95°C for 2 min, 50 cycles at 94°C for 30 s, sity. Detailed clinical records were available for all 58°C (60°C for rs3733197) for 30 s and 72°C for patients, including age, gender, disease onset time and 30 s. PCR products were heated to 95°C followed by family disease history. Psoriasis area severity index rapid cooling to 40°C to facilitate heteroduplex forma- (PASI) was calculated for every patient. Patients were tion. Melting curve analysis was performed by raising considered to have ‘early-onset’ psoriasis if the onset temperature from 55 to 89°C at 0.2°C ⁄ s. Genotypes of the disease was at any age younger than 40 years were identified by the melting temperatures indicated and ‘late-onset’ psoriasis if the disease shows up later by peaks on the derivate plots. than age of 40. The control group comprised 317 healthy individuals selected from blood bank donors Statistical analysis to maximize the match for age (185 men ⁄ 132 women, mean age: 37.16 ± 9.88 years), gender and geographic The statistical analysis was performed on SHEsis online origin. This study was approved by the Ethics Com- software (http://analysis.bio-x.cn/myAnalysis.php). The mittee of Shenzhen Hospital Peking University and Hardy–Weinberg equilibrium of the BANK 1 polymor- conducted in accordance with the declaration of Hel- phism was examined by chi-square test. The differences sinki guidelines for ethics in research. All patients gave in genotype and allele frequencies between patients and written informed consent for genetic studies. Charac- controls were also compared, and value of P < 0.05 was teristics of the study participants are shown in considered to be significant. Differences in allele fre- Table 1. quency was quantified by odds ratios (OR) and 95% CI. BANK 1 single-nucleotide polymorphisms (SNPs) Power calculation genotyping Power was calculated with the ‘Generic Chi-square Genomic DNA was extracted from fresh peripheral test’ module of the ‘Java Applets for Power and Sam- blood. We selected three functional BANK1 SNPs: ple Size’ software (Lenth, 2007) (http://www.divms. rs10516487 (C>T), rs3733197 (G>A) and rs17266594 uiowa.edu/~rlenth/Power/) at the level of significance (T>C). Genotyping of BANK1 SNPs was carried out where a = 0.05, under the prototype data previously by unlabelled probe high-resolution melting (HRM) identified for SLE (Kozyrev et al., 2008). It was con- assay. Probes are C3-blocked on the 3¢-end to prevent firmed that the sample size we chose could provide extension. Sequences of primer and unlabelled probe sufficient power (>80%) to identify a genetic associa- are shown in Table 2. tion with the three polymorphisms. Unlabelled probe HRM analysis was carried out through asymmetric PCR. After asymmetric PCR, a Results large number of superfluous single strand will com- bined with unlabelled probe. As the temperature High-resolution melting analysis with unlabelled probe drops, it will produce two types of melting curve. The part of curve in low melting temperature (Tm) repre- High-resolution melting analysis with unlabelled C3- sents the region of probe and product. The asymmetric blocked probe was used for genotyping. The sensitivity PCR requires five to ten more cycles than conventional and accuracy of HRM were dramatically improved Table 1. Characteristics of study participants Early-onset Late-onset Mild Moderate Severe psoriasis psoriasis psoriasis <10 psoriasis 10–20 psoriasis >20 Controls Total number of subjects 203 39 23 98 121 317 Men, n (%) 126 (62.1) 32 (82.1) 12 (52.2) 62 (63.3) 84 (69.4) 185 (58.4) Women, n (%) 77 (37.9) 7 (17.9) 11 (47.8) 36 (36.7) 37 (30.6) 132 (41.6) Age at enrolment (years), mean (SD) 30.5 (8.9) 55.6 (9.0) 30.8 (8.9) 33.7 (12.7) 35.6 (13.6) 37.2 (9.9) Age at onset of symptoms (years), mean (SD) 23.7 (7.1) 50.1 (8.2) 23.8 (10.8) 28.6 (11.9) 28.2 (12.5) ª 2011 Blackwell Publishing Ltd, International Journal of Immunogenetics 38, 507–512 Association of BANK1 with psoriasis 509 Table 2. Sequences of primer and unlabelled probe bution for any BANK1 SNPs (rs10516487, rs3733197 and rs17266594) between patients and healthy con- Primer trols. The allele and genotype frequencies are shown SNP name Sequence (5¢–3¢) in Table 3.