In the present study we explored the SLE association of ten promising genes, each of which showed a False Discovery Rate (FDR) of <0.8 in our previous TDT-based study. The candidate genes evaluated in the present study are BCL6, CASP10, IL-16, IRF5, KIR2DS4, KLRG1, PRL, PTPN22, PTPRT and TLR8. The present case-control study included an independent childhood-onset cohort of 769 childhood-onset SLE and 5337 cases of adult-onset SLE subjects and 5317 healthy controls each being composed of four ethnicities, as detailed in Supplementary Table 1
An important component of our approach was the deliberate recruitment and usage of childhood-onset SLE cases. They present a unique subgroup of patients for genetic study because their earlier disease onset, a more severe disease course, a greater frequency of family history of SLE, and a lesser effect of sex hormones in disease development (12
) implies a higher genetic load or a more penetrant expression of this genetic load. However, because childhood-onset SLE may also show the involvement of different genetic factors relative to adult onset disease, we analyzed childhood-onset and adult-onset groups of SLE patients separately.
In order to account for any potential confounding substructure or admixture we performed principal component analyses (PCA) (14
) as detailed in Patients and Methods
. Excluding the outliers identified by PCA resulted in low inflation factors in all ethnicities except Hispanic Americans, with only the latter requiring additional PC correction.
As we are performing tests of multiple related hypotheses, controlling for study-wide significance is an important concern to avoid promulgating false positives due to the multiple testing. A classical correction for multiple testing is the Bonferroni correction (or similar family-wise error rate corrections). Unfortunately, it is both too strict and inappropriate in studies such as the present one because of the underlying assumption that each test is independent, whereas in actuality a complex and unknown interdependence is present among SNPs in linkage disequilibrium (11
). In light of this, we have instead calculated an estimate of the false discovery rate (FDR), which measures the number of false positives (Type I errors) we would have to accept to consider a result a true discovery (reject the null hypothesis), using the Benjamini and Hochberg procedure (16
), considering the total number of SNPs tested and the four different ethnic groups (Supplementary Table 1
). Combined p values were calculated from the per-ethnicity p value using the Fisher method. shows that 28 SNPs from 7 genes out of the 10 tested have significant combined association with SLE in adult or childhood-onset subgroups after correction for multiple testing. The complete data on all SNPs tested in this study are given in Supplementary Table 2
. Importantly, these genes include the previously associated genes PTPN22 and IRF5 but also several novel genes that have not yet been associated with SLE. We did not find significant association with the SNPs genotyped in KIR2DS4, PRL and BCL6 in either childhood- or adult-onset SLE. With the exception of rs2476601 in PTPN22, none of the SNPs which we found to be significant code for amino acid changes; only rs11073001 in IL-16 is in an exon, but this variant does not encode for a different amino acid. The most significant SNP found was rs4728142 in IRF5, with a combined p value in adults on the order of 10−29
, and a corresponding FDR on the order of 10−27
Table 1 Significant SNPs which are associated with adult- and childhood-onset SLE as determined by case-control study. Combined p values of adult- and childhood-onset SLE were calculated using the Fisher method and combined False Discovery Rate (FDR) multiple (more ...)
shows the association of SNPs from four novel genes: KLRG1, IL-16, PTPRT and TLR8 with SLE in four ethnic groups (European Americans [EA], African Americans [AA], Asian Americans [AsA], and Hispanic Americans [HA]) in childhood-and adult-onset SLE cases. It is noteworthy that the majority of the significantly associated SNPs show significance in multiple ethnicities both in adult-onset and in childhood-onset SLE. Nevertheless it is also important to notice cases where SLE association is strongly ethnicity-dependent. For example, the SNPs around exon 1 of TLR8 are not significant in AsA but are significant in HA both in children and adults. These graphs also show the distribution of significant SNPs in the genes. For example, the significant SNPs in IL-16 are concentrated around exon 18.
Figure 1 Association of SNPs in KLRG1, IL-16, PTPRT, and TLR8 with SLE in four ethnic groups (European Americans, EA; African Americans, AA; Asian Americans, AsA; and Hispanic Americans, HA) in childhood-and adult-onset SLE cases. The position of exons (green (more ...)
Next we performed haplotype analyses in different ethnic groups, children and adults separately (, , and Supplementary Tables 3
). Supplementary Table 3
depicts the significant haplotype blocks in KLRG1 which are noticeably different in the various ethnicities. Interestingly no significant haplotype blocks were found in adult EA. The significant haplotype blocks in IL-16 are limited to childhood-onset HA (Supplementary Table 4
). As shown in Supplementary Table 5
, the significant haplotype blocks in PTPRT were limited to AsA and a smaller block in childhood-onset HA.
IRF5 haplotype block associated with SLE with p<0.05. SNPs are identified by their refSNP ID.
TLR8 haplotype block associated with SLE with p<0.05. SNPs are identified by their refSNP ID.
BCL6 haplotype block associated with SLE with p<0.05. SNPs are identified by their refSNP ID.
IRF5 has a large number of significant haplotype blocks which are similar in the various ethnicities beside in AA ().Comparing our results with the previously published data on IRF5 association with SLE, we found that rs729302 SNP was reported to be associated with SLE in an EA population with a p-value of 4×10−04
), or p-value of 5.2×10−7
), in Swedish cohort with a p-value of 2.7×10−4
(not corrected for multitest)(19
) and in family trios (uncorrected p-value of 5.0×10−4
). We confirmed these findings on an EA cohort with a multitest corrected FDR of 3.4×10−9
in adults and 1.8×10−8
in childhood-onset cases as shown in . Furthermore, we found significant association of this rs729302 SNP with SLE in HA adults (q value of 8.0×10−3
()), and combined children (FDR of 1.6×10−5
(), but not in AA or AsA cohorts in either adult- or childhood-onset SLE (). The previously reported association of rs4728142 in a Swedish cohort (19
) and family trios (20
) was confirmed by us and extended to all four ethnicities in adult-onset, and to all ethnicities, except AA in childhood-onset disease (). We have also confirmed the involvement of rs2004640 in EA (17
), African Americans (18
), Chinese (21
) and family trios (20
), and in both childhood- and adult-onset SLE in each ethnicity, except for childhood-onset HA (). Association with rs752637 in Europeans was demonstrated by some previous investigators (18
) but not by others (17
). Our studies found a strong association of this SNP with SLE in EA adults (FDR 1.4×10−10
), but not as strong in adult HA, AsA or children EA cohorts ().
Significant SNPs in IRF5 with frequencies, odds ratios, p-values and FDRs given per ethnicity separately for adult- and childhood-onset SLE
We have confirmed the previous association of rs3807306 with SLE demonstrated in a European cohort (19
) and in EA and AA (18
) and extended this association to HA and AsA (). The association of rs3807306 in AA was not significant in our study (q value 0.09), but with an uncorrected p-value of 0.03, it does not contradict the results of a previous study (18
). Also, in agreement with Sigurdsson et al., (19
) we did not detect association of rs1874328 with SLE (Supplementary Table 2
). Underscoring the ethnic dependence of many SNP associations, rs3807135, previously found to be SLE-associated in a family trio study (20
), was found by us to be associated in adults only in EA and HA, but not in AsA or AA, with a very low q value of 0.51 for adult-onset AA (). We have also confirmed SLE-associated haplotype block in the same region as reported previously (17
) and extended this block in chromosome region and detected its SLE association with other ethnicities ().
shows significant haplotype blocks in TLR8 which are distributed throughout the gene, though childhood-onset EA has a haplotype with much higher significance located in the 5′UTR of TLR8. In addition, shows the LD between SNPs that compose the haplotype blocks of TLR8 in adult-onset AA (panel A) and childhood-onset AA (panel B) and EA (panel C). These panels illustrate the differences in LD structure which lead to distinct haplotype blocks observed in different ethnicities.
Figure 2 Schematic representation of multiple significant haplotype blocks in TLR8 found in adultonset AA, childhood-onset AA, and childhood-onset EA. Blocks connecting SNP pairs are shaded according to the strength of the linkage disequilibrium as measured by (more ...)
Although no SNPs found in BCL6 survived the multitest correction, the haplotype analysis indicated that a haplotype block in childhood-onset AsA is significantly associated with the disease () underlining the utility of haplotype analysis even in the absence of singly significant SNPs. The LD structure which led to this haplotype block is depicted in .
Figure 3 Schematic representation of three haplotype blocks in BCL6 found in childhood-onset AsA. Blocks connecting SNP pairs are shaded according to the strength of the linkage disequilibrium as measured by D’ from 0 (white) to 1 (red). Haplotype blocks (more ...)