Importantly, our lymphoblastoid cell model findings recapitulated clinical observations that Asian individuals are particularly sensitive to platinum agents compared with their international counterparts. Using the principle, then, that a population enriched for a given phenotype is ideal for the study of genetic variants responsible for that phenotype, we were able to identify a novel list of SNPs and related genes associated with platinum susceptibility in ASN. The SNPs comprised highly descriptive genetic signatures of susceptibility in ASN, explaining more than 95% of the observed variability in cisplatin susceptibility, and more than 75% of the variation in carboplatin susceptibility. The final-model SNPs were also further validated by demonstrating their association with the differential expression of several target genes in ASN (for each drug) which themselves were shown to be linearly correlated with platinum-induced cytotoxicity in ASN. Finally, several ASN-derived SNPs were also shown to be associated with platinum susceptibility in an independent Caucasian/African population, indicating that, in addition to population-specific signatures, our method was able to identify variants that may be important across populations.
In considering the means by which the SNPs of interest might govern platinum susceptibility, our analysis of genes related to these SNPs produced several interesting findings. From the standpoint of target genes whose expression was associated with the SNPs of interest, the most interesting finding was that one gene family, histone H3, was identified as a key target for both cisplatin and carboplatin, suggesting a platinum drug class effect. Given the function of histones as essential parts of the nucleosome, the evidence that histone-free DNA favors platinum adduct formation [25
], and the fact that nucleosome disassembly must occur for platinum lesions to be repaired [26
], it is especially interesting that our genome-wide approach identified histone H3 as a consistent, strong finding. Others have previously shown that platinums also form adducts on histones – specifically with histone H3 for cisplatin – and have suggested that such histone adducts could act as a nuclear `reservoir' of platinum compounds for further DNA adduct formation [27
]. Wang and Lippard [28
] showed that cisplatin treatment results in histone H3 phosphorylation in cancer cells. To our knowledge, our findings are the first to suggest that specific genetic variants might mediate these mechanisms in a pharmacogenetic manner.
From the standpoint of `host genes' for the SNPs of interest, six unique SNPs within CAMTA1
were associated with carboplatin susceptibility in the cross-population analysis of our study. CAMTA1
encodes Homo sapiens calmodulin binding transcription activator 1, a transcription factor that may participate in the induction of cell differentiation and cell cycle regulation [29
]. In the arena of oncology, it is noteworthy that CAMTA1
is frequently deleted in human neuroblastomas, and low expression of CAMTA1
is related to worse outcomes [30
]. For cisplatin, it was interesting to find a significant SNP in the gene GALNTL4
, as this gene was earlier identified as associated with cisplatin susceptibility by our laboratory using LCLs derived from YRI samples and a modified method of analysis [11
]. Even more intriguingly, the cross-population cisplatin-identified SNP in GALNTL4
– rs7937567 – was associated with the differential expression of the target gene LY86
, which was one of the genes that survived the rigorous three-step association analysis with rs2309997 in ASN. These two SNPs are not in LD. While not only heightening the potential importance of LY86
(lymphocyte antigen 86, which itself has been found to be uniquely upregulated in tumor-associated macrophages compared to normal macrophages [31
]), this finding suggests that an SNP like rs7937567, which is apparently important across multiple populations, may, in ASN, coregulate the common target gene LY86
along with another SNP (rs2309997) thereby providing biological pliability and a potential basis for allelic dose regulation of the phenotype.
We compared our current SNP findings to the results we reported earlier using linkage-directed association studies of these two drugs [5
]. Although the prior studies included only Caucasians, because this is the only population that LCLs from pedigrees are available, there is some interesting overlap. For carboplatin, rs7102746 () falls under the previously identified linkage peak on chromosome 11 with the 1-logarithm of odds (LOD) confidence interval of 11q21-q23.1 (95–105 cm, peak at 99 cm) having a peak LOD score of genome-wide significance of 3.36 (for the IC50
]. This SNP was not interrogated in subsequent linkage-directed association studies because those studies were limited to SNPs within genes. In addition, all of the currently identified SNPs in CAMTA1
() fall under the previously identified linkage peak on chromosome 1 with the 1-LOD confidence interval 1p36.32-36.22 (6–23 cm, peak at 15 cm) with a peak at LOD 1.64 for the 10 μmol/l carboplatin phenotype [5
]. The current novel carboplatin SNPs in CAMTA1
fall very close to the peak of this region. In our earlier publication, we performed linkage analysis on all drug concentrations but followed up with association studies on only linkage regions associated with the IC50
For cisplatin, one current SNP (rs1520896) () interestingly falls under two previously identified cisplatin linkage peaks (also on chromosome 11, 11p15.4-q13.2), which had peak LOD scores of 2.15 (5 μmol/l cisplatin phenotype, 1-LOD confidence interval 11–67 cm, peak at 22 cm) and 2.50 (10 μmol/l cisplatin phenotype, 1-LOD confidence interval 13–55 cm, peak at 22 cm) [32
]. This SNP did not meet the P
significance threshold in a linkage-directed association study of cisplatin IC50
phenotype in 86 CEU samples [32
It is important to note that the individual SNPs identified in our genome-wide approach may not themselves be the causative SNPs governing the phenotype relationship. As the identified SNPs were all genotyped as part of the HapMap project, it is equally possible that any SNP in LD with the identified SNP is the causative locus. This does not, however, affect the translational applicability of our findings, as clinical genotyping of any SNP in perfect LD with the causative SNP should be adequate; however, the possibility exists that a rarer SNP in LD may have an even larger effect. In addition, each individual's specific platinum susceptibility phenotype is likely to be a composite of the risk allele effects for some or all of the identified SNPs (in addition to other undiscovered SNPs), explaining why, upon inspection of the data from and , some individuals homozygous for one apparent `risk' allele, for example, can have relatively lower platinum susceptibility (higher IC50
s) than other individuals who are heterozygous or absent for that same given allele. In other words, rather than functioning alone to determine the phenotype, each SNP of interest acts in concert with the other genetic determinants to determine phenotype in a given individual. This concept has been advanced since other studies of genetic susceptibility that assess or test the effects of one SNP in isolation often reveal a very low odds-ratios of effect on the phenotype, or no replicable effect at all, but studies which instead consider complex trait genetics as determined by a genetic signature (or haplotype) of multiple interacting SNPs have found that relatively large odds ratios of effect can be found, even when any one SNP by itself is not found to be significant [33
]. We believe that this concept is likely also true of the multiple SNPs of interest implicated by this work. Our backwards elimination predictive model supports this statement, showing the robust ability with which a group of SNPs (a genetic signature) can describe the overall phenotypic variation.
Several candidate genes implicated by other earlier studies of platinum toxicity susceptibility, such as ERCC1
, and EPHA2
], were not identified using our model, but this may be because of characteristics of the gene expression profile of lympho-blasts, limited HapMap genotyping coverage of previously-implicated candidate genes, relatively modest (yet still important) statistical association in the initial ASN GWAS, or because these genes are not important in Asians, from which all findings in our model originated. These considerations emphasize the paramount relevance of combining our genome-wide method's results with those of candidate gene methods to arrive at a composite picture of platinum susceptibility. In addition, it should be emphasized that there may be critical rare variants in the ASN population (having a MAF <5%) that would not have been detected by this study because of the method we used. Such SNPs, in fact, may be most important toward explaining interethnic susceptibility differences [35
]. Our cross-population SNP-identifying method, however, offers the advantage of identifying relatively common SNPs testable in individuals of diverse ethnicities. Restriction of our analysis to identify the uncommon variants might also be fruitful and could represent a novel model for investigating genetic variation underlying ethnic differences in response to chemotherapy drugs [36
One limitation of our work is that, on account of the relatively small sizes of our population cohorts, the chosen P
value cut-offs for the initial ASN GWAS and for cross-population significance testing, when corrected for multiple testing, would not meet the most stringent levels of significance [38
]. Although it remains possible that some or all of the SNP associations are produced by chance alone, we believe that this possibility is highly unlikely given the facts that (a) the SNPs of interest, when tested in multi-SNP genetic signatures (which more closely approximate the biology) rather than in isolation, are highly significant in backwards elimination models which show that the genetic contribution of these signatures on predicting platinum susceptibility is robust; (b) the finding of multiple, independently identified SNPs within the same host gene (as with the SNPs identified in CAMTA1
for carboplatin) suggests that this location is much less likely to be identified by chance, but rather, adds import to this region as a true region of significance for susceptibility to the drug; (c) several of our SNPs fall under previously identified linkage regions associated with susceptibility to these drugs; and (d) target gene expression analysis for the SNPs shows that several are strong master regulators of multiple genes, that these genes themselves are independently correlated to platinum sensitivity, and that one gene is implicated for both cisplatin and carboplatin showing an expected commonality within the platinum family. Separately, our approach defined ASN as the combined Chinese and Japanese HapMap populations – a method proposed by the plan of the International HapMap project [13
], supported by genetic ancestry/migration data on these populations [39
], and consistent with earlier data suggesting significant genetic homogeneity [40
] – but it is still likely that some genetic differences exist between these two populations [41
]. Finally, these cell-based studies of human LCLs are intended to allow discovery of SNPs which can then be tested in a formal clinical trial, following the paradigm set forth by Hirschhorn and Daly [42
]. Such clinical testing is currently ongoing.
In summary, we used a cell-based, genome-wide approach centered on a phenotypically enriched population to identify novel genetic variants governing cisplatin and carboplatin susceptibility. The composite list of novel SNPs and related genes, along with SNPs in previously identified platinum pathway genes, deserve study in clinical settings toward the goal of identifying individuals at risk of toxicity from, and perhaps response to, these commonly used platinum agents.