This is the first study, to our knowledge, that performed high-density PAI studies of a human ortholgous locus for a mouse tumor susceptibility region. These data confirm our previous studies in which markers on chromosome 7 demonstrated PAI 12
and complement the mouse linkage studies 6-8
. Our data showed eight HDAC9
SNPs with suggestive evidence and one HDAC9
SNP with statistically significant evidence for PAI in cSCCs, indicating these variants or those they tag for could represent variants important in the development or progression of cSCC.
Utilizing previous aCGH and microsatellite data, we identified 15-22% of tumors demonstrating copy-neutral LOH at SKTS5
which provides additional support for this region as being important in cSCC development. LOH is commonly evaluated by measuring changes in copy number; however LOH can also occur independently of copy number change. Copy-neutral LOH has been observed in multiple types of cancers 15,16
. Studies that use copy number changes to identify candidate loci may overlook regions with high levels of copy-neutral LOH. Here, we correlated aCGH and microsatellite genotyping data. We identified 9 of 59 tumors with copy-neutral LOH at SKTS5
, suggesting a higher frequency of imbalance at this locus than previously appreciated and increasing the potential relevance of this locus in cSCC.
This study identified nine SNPs within HDAC9
which demonstrated PAI in cSCCs, suggesting this gene may play an important role in skin cancer. HDAC9 is a class IIa histone deacetylase family member and is thought to regulate the epigenetic status of histones by catalyzing deacetylation. In the strains of mice used for mapping Skts5
, we identified both potentially functional amino acids and differential expression of Hdac9 8
. Aberrant HDAC9
expression is observed in several types of cancers including medulloblastoma 17
, acute lymphoblastic leukemia 18
, and cervical carcinoma 19
has non-histone protein targets including forkhead box protein 3 20
, ataxia telangiectasia group D-complementing protein 21
, and glioblastoma 1 protein 22
; which are members of pathways implicated in tumorigenesis 21,23
. Inhibition of HDAC9
also inhibits cellular proliferation and induces apoptosis 21, 22
. Genomewide association studies have identified SNPs in HDAC9
that are associated with male-pattern baldness 24
. Taken together, these studies suggest a link between HDAC9
, the skin/hair follicle and cancer-related phenotypes.
Alleles showing preferential gain in tumors or those that they tag for may be strong candidates for risk association studies. Gain of specific alleles in tumors may indicate growth or selective advantage of cells containing these alleles. We do not yet know which SNP is the “causal” SNP driving the observed PAI. The nine HDAC9 SNPs showing PAI in tumors are intronic, as are the majority of SNPs for which that they tag. If the causal SNP is one from our study, they may be influencing splicing, regulation through enhancer activity or noncoding RNA, or affecting the methylation status of HDAC9. Preferential gain of HDAC9 variants in tumors is suggestive that the gained alleles may act to promote cancer phenotypes. These variants may induce stronger expression and/or activity of HDAC9, as both characteristics have been linked to tumorigenesis.
There are limitations to this study. We only assessed variants from a limited number of genes at SKTS5 and may have missed the causal gene or variants. Genes were prioritized based on the mouse data, but only two genes were represented by multiple tagging SNPs. We have strong coverage for both AHR and HDAC9, although not all SNPs or haplotypes may be fully represented. Because we only analyzed one tagging SNP in the other genes, these genes were not comprehensively evaluated. We also only chose SNPs with a high degree of heterozygosity as we were not adequately powered to detect preferential allelic imbalance for variants with low heterozygosity frequencies; thus this study is underpowered for the detection of rarer SNPs. Another important consideration is that only one of the SNPs showing evidence for preferential allelic imbalance met multiple comparisons adjustments for the 108 SNPs evaluated. However, as many of these SNPs are highly correlated and are in linkage disequilibrium, a conservative Bonferroni correction may not have been an appropriate method for adjustment. It is possible that the SNPs identified in this study are playing a role in tumorigenesis and may be somatic targets. Future studies will focus on identifying the causal SNP for the observed PAI and functional studies in vitro to characterize the variants driving imbalance and their potential role in cancer initiation and progression.
In summary, this study highlights the importance of investigating copy-neutral LOH to identify loci critical for tumor development. Furthermore, our data support the use of cross-species strategies to identify candidate genes. We identified HDAC9 as a candidate gene for human cSCC using a combination of linkage mapping in the mouse with targeted PAI mapping in human tumors. Although there is strong evidence for PAI for SKTS5 and HDAC9, additional functional, genetic, and population-based studies are necessary to follow up on these findings.