Discovering genes involved in severe cutaneous adverse reactions and especially SJS/TEN is a major challenge for pharmacogenetics as these reactions, when not fatal, are a sword of Damocles for those who already had the disease and fear to take any drug. Despite important efforts, only genes located in the HLA region have been identified so far. One possible explanation for this lack of success is the limited sample sizes of patients available. Indeed SJS/TEN is fortunately a very rare disease and in most studies, sample sizes rarely exceed one or two hundreds of patients, making it difficult to investigate more than a few candidate genes. Through a collaborative effort, the RegiSCAR group was able to collect detailed medical information and DNA of more than half a thousand of patients from Europe who were genotyped on Illumina 317 K chips. By comparing their genotypes to the ones of 1,881 controls genetically matched for the country of origin, we were able to study the SJS/TEN association at a genome-wide level on European samples. Apart from six SNPs located in the HLA region, no other locus was found to be associated with the disease at a high enough significance level to ensure it is not false positive due to the multiple tests performed. Sample sizes of half a thousand are not enough to ensure a good power to detect common variants with small effects (OR in the range between 1.1 and 1.3) similar to those identified in several common diseases such as diabetes or different cancers  where larger samples are easier to collect. However, given the 424 patients remaining after Quality Control, the study was powerful enough to detect common variants with modest effects (the power exceeded 80% to detect variants with an allele frequency above 15% conferring ORs above 1.7 under a multiplicative model). The fact that only the HLA region is detected, suggests that there might not be any other common variant that confer a substantial increase in disease risk and could thus be of interest as a predictive factor. These power computations might however be too optimistic as they are based on the assumption that the same genetic variants might be involved in cases of SJS/TEN associated to different drugs. If we consider that there might exist some levels of genetic heterogeneity depending on the drugs involved as it is the case for HLA associations, the power can be dramatically reduced. For example, considering only the 57 allopurinol-exposed cases, the power to detect a variant of frequency 15% conferring an OR of 2 is then only 5% at a nominal type-one error rate value of 10-6. On the other hand, the power to detect a similar effect than the one observed at rs9469003 (i.e., risk allele of frequency of 15% with an associated OR of ~4 among allopurinol-induced SJS/TEN) is 99%, showing that the study was not underpowered to detect strong effects even if they were restricted to a small subset of patients.
It is true however that we might have missed some variants with important effects, especially those with minor allele frequencies below 5% that are not well covered by SNP-chips and it could thus be of interest to investigate the association with rare variants. If the "common disease-common variant hypothesis" was believed to explain the susceptibility to multifactorial diseases [30, 31], results from GWAS have shown that common variants only explain a minor part of most common disease heritabilities. It has been suggested that rare variants could explain at least part of this "missing heritability" and indeed, rare variants have been found in several diseases [32–36]. Interestingly these rare variants are often functional variants with a direct impact on the protein functionality and they usually confer a much stronger increase in disease risk than common variants. They are also more likely to be affected by some moderate levels of negative selection [37, 38]. For diseases such as adverse drug reactions, it is not unlikely that the genes and variants involved could be under selection. Indeed if most of the drugs have been introduced too recently to be directly responsible for selective pressures, they are often derived from nutrients that have been consumed by humans for a long time. Thus, genes involved in drug metabolism can potentially be involved in natural selection and this was confirmed by previous studies [39–41]. However, the implication of drug metabolism genes in SJS/TEN remains to be established.
Concerning the association with HLA detected in this sample, we confirmed that it is drug-specific with the strongest association found for the group of patients where allopurinol is suspected to be the cause of the disease. However the disease association was still detectable after exclusion of all patients exposed to allopurinol (whether considered as causing the reaction or not) (OR = 2.13, 95% CI = [1.41; 3.23]). This suggests that different HLA alleles are probably involved depending on the drug, and the 6 SNPs identified could be those that were in linkage disequilibrium (LD) with several of these HLA alleles. To further explore this hypothesis, we tried to impute HLA-B genotypes from the SNP data  but we were not successful because of the presence of multiple rare alleles at this locus such as HLA-B58 itself that is only present in one of the European families genotyped by de Bakker et al.  precluding the possibility to find appropriate tags for this allele using this sample. To overcome the problem, we tried to determine tag SNPs using our own HLA-B genotyped sample that was enriched in HLA-B58 but we did not find any SNP or group of SNPs that was correlated enough with the HLA-B58 allele (the maximum r2 value was below 20%). This poor HLA-B tagging ability of the SNPs could certainly explain why the association found here in relation with allopurinol is much weaker than the one reported in studies on European samples using resolved HLA-B alleles [16, 44]. However, even in these two latter studies, the association with HLA-B*5801 is much weaker than the one found in Han Chinese  or Thai populations  where all patients with allopurinol-induced SJS/TEN are carriers of this allele. It is thus possible that the HLA genetic determinants involved in SJS/TEN are not the HLA-B alleles themselves but some loci in LD with them. Further studies in this genomic region would certainly be necessary to better understand the mechanisms involved.
On a methodological point of view, this study illustrates how Reference Control Panels could be used to test for association when only cases are genotyped. Only patients were genotyped here and controls were selected from the CNG European Control Panel  based on their country of origin that was either France and Germany, since those were the two main countries where the cases come from. We also tried to use the whole panel of controls and correct for population stratification using Principal Component Analysis (PCA) and an adjustment on up to 20 PCs instead of the 2 used here. We found that including controls that were more genetically distant to the cases does not really improve the signal in the HLA region and instead could lead to false positive results in other genomic regions. This is somewhat in contradiction with the results recently reported by Zhuang et al.  and will probably need to be further investigated. We also used some alternative approaches where controls were genetically matched to cases using either a distance based on the identity by state as described by Guan et al.  or a distance derived from the top PCs of the PCA performed to identify axes of variations similar to the one proposed by Luca et al. . Tests accounting for the matching units were then performed and we found that only the HLA region pointed out with significance levels in the same order of magnitude as the ones obtained here. These matching strategies might however be of interest as they could allow the analysis of more heterogeneous samples by including for example those non-European RegiSCAR cases that were excluded here and matching them to individuals from the CEPH-Human Genome Diversity Panel .