Association of single nucleotide polymorphisms ( SNPs ) of PADI 4 gene with rheumatoid arthritis ( RA ) in Indian population

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease affecting ~ 1% of the population worldwide. The genome wide association studies on RA patients revealed linkage with 1p36 locus containing peptidyl arginine deiminase 4 (PADI4) genes. Case-control association studies and mRNA stability assays reported the association of PADI4 gene with RA in Korean and Japanese populations. However, such association was not found in Spanish population. Differences in the association of PADI4 with RA in different populations prompted the present study in Indian population. Anti-CCP antibodies, RF antibody, disease activity scores at 28 joints (DAS28) and mutations in three exons of PADI4 were investigated in RA patients and control group. Among the patients antiCCP antibody levels were found to be associated with high DAS28 values (r = 0.4526, P < 0.0001). Polymorphism in exon-4 (padi4_104, [rs1748033]) of PADI4 showed significant association of ‘C’ allele with RA in the study population (P = 0.0008). Polymorphism in exon-3 (padi4_92, [rs874881]) also exhibited moderate association with the disease (P = 0.075). However, no association of the disease was found with the SNPs padi4_89 [rs11203366] and padi4_90 [rs11203367] in exon-2 of PADI4.


INTRODUCTION
Rheumatoid arthritis (RA) is a complex autoimmune disease which affects about 0.5 -1% of the human population worldwide (Hochberg and Spector, 1990).Besides environmental factors, genetic factors are believed to be responsible for ~ 60% of the risk of developing RA (MacGregor et al., 2000).HLA locus accounts for approximately one-third of genetic susceptibility to the disease (Deighton et al., 1989).Besides HLA a number of other candidate genes have been investigated (Barton et al., 2004a(Barton et al., , 2004b;;Fan et al., 2008;Chen et al., 2010), but unfortunately, no common loci have been identified.The genome wide study of RA sibling pairs revealed linkage with the 1p36 locus containing all peptidyl arginine deiminase (PADI) genes (Cornelis et al., 1998).PADI enzymes convert the peptidyl arginine into peptidyl citrulline by post translational deimination.In Japanese population, a case-control association study using single nucleotide polymorphisms (SNPs) identified a haplotype-associated susceptibility to RA in PADI4 gene (Suzuki et al., 2003;Utz et al., 2004).Correlation was observed between the PADI4 gene and RA in the French population (Gandjbakhch et al., 2009).mRNA stability analysis revealed association of PADI4 gene with RA in Korean, Japanese and German populations (Suzuki et al., 2003;Hoppe et al., 2006;Kang et al., 2006).However, Spanish and UK populations failed to replicate the association (Barton et al., 2004b;Martinez et al., 2005).Similarly, polymorphisms in PADI4 gene did not contribute to susceptibility in Chinese Han population (Chen et al., 2011).The varied results on association of PADI4 gene SNPs with RA in various populations necessitated the present investigation in Indian population.For decades, the classification of RA relied mainly on the criteria described by the American College of Rheumatology (ACR) (Arnett et al., 1988), wherein a single serological marker, 'rheumatoid factor' (RF), has been included.However, the presence of RF is not restricted to RA patients, and can also be detected in patients suffering from other diseases like chronic hepatitis C infection (Wener et al., 2004), as well as in a percentage of healthy elderly people (van Venrooij et al., 2002), indicating the need for a more specific and more sensitive diagnostic marker.The PADI-mediated post-translational deimination of peptidyl arginine could generate a non-standard aminoacid peptidyl citrulline (Nijenhuis et al., 2004).Antibodies targeting citrullinated epitopes exhibited high sensitivity with superior specificity (van Venrooij et al., 2002) to RA, favouring anti-CCP antibodies as a more reliable RA marker.Though anti-CCP is accepted as a diagnostic marker, yet its association with the disease severity remains unclear (Onder et al., 2009).The present study aims at investigation of the association among SNPs in three exons of PADI4, anti-CCP antibodies, RF levels and disease activity score at 28 joints (DAS28) in RA patients of the Indian population.

MATERIALS AND METHODS
Patients and controls Blood samples were collected from 101 patients at a Rheumatology clinic in Hyderabad, Andhra Pradesh, India.Patients fulfilling at least 4 of the American College of Rheumatology (formerly the American Rheumatism Association) revised criteria for the classification of RA were considered (Arnett et al., 1988).All the 101 patients were tested for ankylosing spondylitis (AS) by employing Polymerase Chain Reaction (PCR) amplification with HLA-B27 specific primers.Six of them positive for AS were eliminated from the study.The remaining 95 patients confirmed for RA were included in the study.Fifty six age matched healthy blood donors (belonging to Andhra Pradesh state) served as controls.It was ensured that all the selected patients and controls are native of Andhra Pradesh state.Further, the same study group (both case and control subjects) was used to analyze the variation of other phosphatase genes (PTPN22 and PTPRC) and it had no allelic variation at these loci indicating that their genetic background is not heterogeneous (unpublished data).However, there may be a possibility of small background differences which was not assessed in this study.Clinical data on age, sex, disease duration and disease activity scores were collected.In addition, immunological tests for anti-cyclic citrullinated peptide antibodies and Rheumatoid Factor (IgM) antibodies were conducted.All patients gave written informed consent and the Ethics Committee of the Institute approved this study.Clinical characteristics of the patients are indicated in Table 1.

Detection of anti-CCP antibodies A commercially available,
second-generation anti-CCP ELISA (Immunoscan RA, Euro-Diagnostica, Sweden) was used for the quantification of anti-CCP antibodies.Individuals with ≥ 25 IU/ml were regarded as anti-CCP antibody positive.Antigen coated wells were applied with calibrators, controls and sera of the subjects.After incubation, the wells were washed with wash buffer, then incubated with the conjugate solution containing horse radish peroxidase (HRP) labeled anti-human IgG and washed with wash buffer.Later, substrate solution was added and the reaction was stopped.Absorbance values were read immediately at 450 nm.
Rheumatoid factor determination IgM RF values were determined by Enzyme Linked Immuno Sorbent assay according to the manufacturer's instructions (Euro-Diagnostica, Sweden).Individuals with values ≥ 20 IU/ml were regarded as RF positive.Antigen coated wells were incubated with calibrators, sera of subjects followed by washing with wash buffer.Then incubated with antihuman horse radish peroxidase conjugate solution and excess antibodies were removed with wash buffer.Later, chromogenic substrate solution was added and the reaction was stopped.Absorbance values were recorded immediately at 450 nm.
Extraction of DNA Genomic DNA was extracted from EDTA-treated frozen whole blood by a rapid non-enzymatic method (Lahiri and Nurnberger, 1991).EDTA-treated frozen whole blood was mixed with low salt buffer and non-ionic detergent.Nuclear pellet was resuspended in high salt buffer and sodium dodecyl sulphate (SDS), incubated at 55°C.After adding the sodium chloride the supernatant was taken.The DNA was precipitated with 100% alcohol and washed with 70% alcohol.Finally the DNA was resuspended in TE buffer (10 mM TRIS, 1 mM EDTA, pH 8.0).
Calculation of DAS28 values DAS28 was calculated based on the parameters, viz., swollen joint count, tender joint count, erythrocyte sedimentation rate (ESR) and general health, using DAS Calculator.The non-laboratory parameters were recorded as per physician's opinion.

Anti-CCP antibodies and RF antibody
Out of 95 RA patients, 54 (56.8%) showed the presence of anti-CCP antibodies and 74 patients (77.8%) were found positive for RF antibodies.Among patients, 53 were positive for both anti-CCP antibodies and RF antibodies; 22 negative for anti-CCP antibodies and positive for RF antibodies; only one patient was positive for anti-CCP antibodies and negative for RF antibodies; and 19 were negative for both anti-CCP antibodies and RF antibodies.
Genotyping results of exons 2, 3 and 4 of PADI4 are given in Table 2. Analysis of 4 SNPs revealed the association of A-C-C-C haplotype with the disease (Table 3).Except RF negative RA and disease duration less than median RA, the other parameters such as., RF positive RA, Anti-CCP positive RA, Anti-CCP negative RA, DAS28 and disease duration more than median RA demonstrated significant association with the SNP padi4_104 (rs1748033) (Table 4).

DISCUSSION
The diagnostic and prognostic value for detection of anti-CCP antibodies was shown to be specific to RA patients in various populations (Dubucquoi et al., 2004;Vallbracht and Helmke, 2005;Gupta et al., 2009).In the present study, 56.8% of the RA patients were found positive to anti-CCP antibodies while 77.8% were positive to RF antibodies.Unlike earlier studies (Gupta et al., 2009), only 0.05% of RF-negative RA patients were found positive for anti-CCP antibodies in the study group.Thus, anti-CCP antibodies might serve as a better diagnostic marker along with RF in the Indian population.DAS28 is an index that measures the severity of the disease in RA patients.Anti-CCP levels were associated with high scores of DAS28 in the present study population, which is similar to that of a study in the Turkey population (Onder et al., 2009).However, no such association was found in an earlier study in the Turkey population (Serdaroglu et al., 2008).
PADI enzyme converts the peptidyl arginine to peptidyl citrulline (Vossenaar et al., 2003).PADI4 with four exonic SNPs was thought to be a candidate gene for RA in Japanese population.Out of them, only padi4_92 and padi4_104 SNPs were shown to be associated with RA (Suzuki et al., 2003).Based on these results, the polymorphisms of three exons, viz., exon-2, exon-3 and exon-4 of PADI4 have been analyzed in the present study.The results revealed that only one SNP in the exon-4 (padi4_104) is strongly associated (P = 0.0008) with RA susceptibility in the Indian population.However, all the 4 SNPs were found associated with the susceptibility to RA in the Korean population (Kang et al., 2006).Suzuki et al. (2003) concluded that the more stable mRNA transcript and in turn more citrullinated proteins have been produced by the susceptible haplotype in PADI4.Earlier studies suggested that the anti-CCP antibodies and the susceptible haplotype of PADI4 serve as markers for susceptibility and severity of the disease (Bas et al., 2003;Meyer et al., 2003).As compared to earlier studies on Spanish, Korean and Japanese populations (Suzuki et al., 2003;Hoppe et al., 2006;Kang et al., 2006), the Indian population exhibited marked differences in the association of PADI4 SNPs with RA, which may be attributed to the differences in the ethnicity (Yamada and Yamamoto, 2007).Apart from the PADI4 various other genes might also play role in the susceptibility and severity of the disease in the Indian population and needs further in-depth investigation.

Table 1 .
Clinical characteristics of patients

Table 2
*Allele 'C' of padi4_104 is associated with the disease.

Table 3 .
Structure and frequency of haplotypes of 4 SNPs in PADI4 gene

Table 4 .
Association of the various parameters of RA with the SNP "rs1748033" of PADI4 gene