Data

The phenotype (NAPHENO) and candidate gene (CANDGENE.DAT) data were provided by the North American Rheumatoid Arthritis Consortium [4] and consist of 839 RA cases from sib-pair families and 855 unrelated controls. SNPs with missing data of 15% or greater were excluded from the analysis, resulting in a total of 17 SNPs from 13 genes (PTPN22, CTLA4, HAVCR1, IBD5, SLC22A4, IL3, IL4, SUMO4, MAP3K71P2, DLG5, CARD15, RUNX1, and MIF) being considered. The missing genotypes were estimated using the proximity calculated by RFs on the training subjects [3].

In this study, we generated two replicate data sets in which the subjects were independent within samples and were dependent across samples. This approach ignores dependencies between the data sets that in general lead to inflation of type I error in the replication set. However, we feel that this approach is justified to simplify the confirmatory analyses using the available data. The data set was split into two replication data sets by randomly selecting a case from each affected sib pair for one of the data sets and the remaining case contributing to the second data set, with the controls almost evenly divided into the two data sets. Data set 1 includes 398 cases and 427 controls, while data set 2 includes 387 cases and 428 controls. It should be noted that due to the relatedness between the replication and the analysis data sets, the confirmation from the second data set should be interpreted with caution.

The data from the genome-wide linkage panel (a total of 5742 SNPs, excluding chromosome Y SNPs) were available for 1998 individuals from families with RA patient (NAILL01–NAILL25). For families with two or more siblings, one was randomly selected from each family for data set 1, and the second subject was then randomly selected from the remaining of samples for data set 2. The singletons were randomly divided into the two data sets. Each of the data sets consists of 740 unrelated subjects. The analysis of the replication data set should be considered exploratory.

The RF method is not particularly efficient at imputing missing data for large number of SNPs. Therefore, we applied an extension of the expectation-maximization (EM) algorithm implemented in HelixTree (Golden Helix Inc., Bozeman, MT) to impute the missing genotypes for the genome-wide linkage data set. In a simulation study, this method achieved outstanding performance of imputation accuracy above 95% with missing rate ranging from 1% to 10% (Sun et al., unpublished data).

Association analysis

The association between each candidate gene SNP and RA status was tested using all samples. Two methods were used for the candidate gene data set to adjust for the degree of relatedness among the affected siblings, a generalized estimating equations (GEE) method with likelihood ratio statistics (SAS 8.2, SAS Institute Inc., Cary, NC, USA, 1999) and Risch and Teng's statistic [5]. Results from the Risch and Teng's statistic and GEE likelihood ratio were consistent so that only results from the GEE analysis are reported. SNPs with an association with RA at p < 0.05 were also analyzed in a multiple SNP model.

For the genome-wide data set, single-SNP associations with RA were assessed using a χ² test when all three genotypes were observed and the least frequent genotype observation was more than five. In situations in which these criteria were not met, Fisher's exact test was used to test the association. The p-values were then used to rank the significance of all SNPs.

Identification of phenotypic subgroups among RA patients

An unsupervised clustering analysis was carried out on the RA cases using the detailed phenotypic data (demographic and disease-related clinical and laboratory variables) to identify clinically distinct subgroups of RA patients. For the purpose of the analysis, the phenotypic variables were dichotomized using a threshold of the 75^th percentile for AgeOnset (≥50), TenderJtCt (≥13), SwollenJtCt (≥13), JAMScore (≥52), SeverityLH (≥5), and SeverityRH (≥5). DRB1_1 and DRB1_2 marker variables were combined and dichotomized on the basis of the presence or absence of RA-associated epitopes (*01 and *04 alleles). Patient self-reported American Rheumatism Association (ARA) sub-scores and the overall ARA score were not included in the analysis. All other clinical measurements, except BMI (51% missing), were used in the analysis.

Clustering analysis was carried out using modified 'Jaccard' coefficient (proportion of non-zero pairs that are similar between subjects) as the distance measure [6]. Multidimensional scaling (MDS) of the distance matrix was used to visualize and identify sub-groups in the patient population. After identifying subgroups of RA patients characterized by clinical and laboratory phenotypes, we further investigated whether the SNPs were associated with the sub-groups. Specifically, we attempted to predict the different subgroups within RA cases using the SNP data from the candidate gene study.

RA classification using RFs

In addition to the conventional association analysis methods that were used in the study to identify RA susceptibility loci, we also used RFs [3]. The method was used for classification or prediction of cases and controls using the SNP data, and five-fold cross-validations (CVs) were used to evaluate classification accuracy. The receiver operating characteristic (ROC) curve was calculated based on the vectors of sensitivity and specificity for each of the five CVs. The values of area under curve (AUC) from the five CVs were averaged to compare the predictive ability and stability.

All of the 17 candidate gene SNPs and the 5742 SNPs from the genome-wide linkage panel were included in the RFs model. RFs were also applied using selected subsets of SNPs as predictor variables. These subsets were selected on the basis of their ranking on the importance measurement implemented in the RFs package. The SNPs that were highly ranked were selected for the RFs model.

All statistical analyses were performed using the R statistical software package (version 2.1.0) from R Project http://www.r-project.org/. The R package for Random Forests (randomForest 4.5.-15) was installed and utilized following the instructions.

Candidate gene analysis of RA status

Using all of the 17 candidate gene SNPs, the predictive ability of the RFs model was estimated by the ROC curve with five-fold cross validation. The low average AUCs of data set 1 and 2 (0.59 and 0.54) indicated poor predictive ability in both data sets. This did not improve by using the three most important SNPs ranked by the RFs model (AUCs = 0.59 for both data sets). Furthermore, SNPs PTPN22*rs2476601 and SUM04*rs237025 are consistently ranked the top two on the variable importance score in both data sets. Interestingly, both SNPs show strong associations in the univariate GEE analysis (Table 1).

Candidate gene analysis of subgroups among RA patients

We investigated whether there are phenotypically distinct subgroups among RA patients. Such subgroups may represent etiologically or genetically distinct entities among RA patients, and therefore consideration of these subgroups in a genetic association study may lead to an increase in the power to identify susceptibility genes.

Figure 1 shows the separation of the two clusters, for which a cluster validity algorithm was employed to determine statistical significance. The empirical distribution of a cluster validity statistic, within-to-between ratio, was computed from 10,000 permutations of the partition vector. The test statistic was defined as the ratio of average within cluster distances to the average between cluster distances for a partition vector. The clusters (subgroups A and B) shown in Figure 1, was calculated to be significant at p-value 0.01. These phenotypic clusters are correlated with a set of clinical measures, such as ARA criteria score [7], which reflect the disease severity.

RFs analysis using genome-wide linkage SNP panel

We examined the predictive ability of RFs using the 5742 genome-wide linkage SNPs. Using the classic training-testing strategy with five-fold CV and the AUC of the ROC curves, we found that the RFs model did not build a reliable prediction model for RA status in either data set 1 or 2. However, when we only used SNPs that were highly ranked on the importance score measure (e.g., the top 500 SNPs), high sensitivities and specificities were consistently obtained in all five CVs for both data set 1 (Figure 2) and 2 (similar results, not shown). The sensitivities and specificities were approximately 90% and 80%, respectively. Figure 3 shows the impact of the number of selected SNP predictors. Using the AUC of the ROC curve to present the predictive ability of each RFs model, we found that the overall predictive ability declined when more than the 500 top ranking SNPs were used to build the model. When compared to the randomly selected SNPs in RF modeling, we concluded that the predictive ability can be significantly improved by selecting the most important SNPs and the improvement was not due to the random effects (Figure 3). When we estimated the most important SNPs only in the training data set for each CV, the improvement of predictive ability was not as obvious as in the full data set. This suggests that the size of the data set may not be sufficient and each training set could not represent the whole data set. Therefore, the best set of predictors identified from each training set would not generalize well across the whole data set.