Germline Variants Impact Somatic Events during Tumorigenesis.

Cancer is characterized by diverse genetic alterations in both germline and somatic genomes that disrupt normal biology and provide a selective advantage to cells during tumorigenesis. Germline and somatic genomes have been extensively studied independently, leading to numerous biological insights. Analyses integrating data from both genomes have identified genetic variants impacting somatic events in tumors, including hotspot driver mutations. Interactions among specific germline variants and somatic events influence cancer subtypes, treatment response, and clinical outcomes. Investigation of these complex interactions is increasing our understanding of aberrant pathways in tumors that may uncover novel therapeutic targets. Here, we review the literature describing the role of germline genetic variants in promoting the selection and generation of specific mutations during tumorigenesis.

Developmental subtypes assessed by DNA methylation-iPLEX forecast the natural history of chronic lymphocytic leukemia.

Alterations in global DNA methylation patterns are a major hallmark of cancer and represent attractive biomarkers for personalized risk stratification. Chronic lymphocytic leukemia (CLL) risk stratification studies typically focus on time to first treatment (TTFT), time to progression (TTP) after treatment, and overall survival (OS). Whereas TTFT risk stratification remains similar over time, TTP and OS have changed dramatically with the introduction of targeted therapies, such as the Bruton tyrosine kinase inhibitor ibrutinib. We have shown that genome-wide DNA methylation patterns in CLL are strongly associated with phenotypic differentiation and patient outcomes. Here, we developed a novel assay, termed methylation-iPLEX (Me-iPLEX), for high-throughput quantification of targeted panels of single cytosine guanine dinucleotides from multiple independent loci. Me-iPLEX was used to classify CLL samples into 1 of 3 known epigenetic subtypes (epitypes). We examined the impact of epitype in 1286 CLL patients from 4 independent cohorts representing a comprehensive view of CLL disease course and therapies. We found that epitype significantly predicted TTFT and OS among newly diagnosed CLL patients. Additionally, epitype predicted TTP and OS with 2 common CLL therapies: chemoimmunotherapy and ibrutinib. Epitype retained significance after stratifying by biologically related biomarkers, immunoglobulin heavy chain mutational status, and ZAP70 expression, as well as other common prognostic markers. Furthermore, among several biological traits enriched between epitypes, we found highly biased immunogenetic features, including IGLV3-21 usage in the poorly characterized intermediate-programmed CLL epitype. In summary, Me-iPLEX is an elegant method to assess epigenetic signatures, including robust classification of CLL epitypes that independently stratify patient risk at diagnosis and time of treatment.

Variants in an Hdac9 intronic enhancer plasmid impact Twist1 expression in vitro.

Skin tumor susceptibility 5 (Skts5) was previously mapped to mouse chromosome 12 through linkage analysis of skin tumor susceptible Mus musculus (NIH/Ola-S) and skin tumor resistant outbred Mus spretus (SPRET/Out-R) mice. Hdac9 was identified as a potential candidate for Skts5 based on conserved non-synonymous sequence variants and expression analyses. Studies by others identified an enhancer in human HDAC9 that correlated with TWIST1 expression. We identified 45 sequence variants between NIH/Ola-S and SPRET/Out-R mice from the orthologous region of the human HDAC9 enhancer. Variants mapping to intron 18 differentially affected luciferase expression in vitro. NIH/Ola-S clones showed an approximate 1.7-fold increased luciferase expression relative to vector alone or the equivalent clones from SPRET/Out-R-R. Furthermore, cells transfected with a portion of the NIH/Ola-S intron induced 2.2-fold increases in Twist1 expression, but the same region from SPRET/Out-R mice resulted in no up-regulation of Twist1. In silico transcription factor analyses identified multiple transcription factors predicted to differentially bind NIH/Ola-S and SPRET/Out-R polymorphic sites. Chromatin immunoprecipitation studies of two transcription factors, Gata3 and Oct1, demonstrated differential binding between NIH/Ola-S and SPRET/Out-R plasmids that corroborated the in silico predictions. Together these studies provide evidence that the murine orthologous region to a human HDAC9 enhancer also acts as a transcriptional enhancer for mouse Twist1. As ectopic sequence variants between NIH/Ola-S and SPRET/Out-R differentially impacted luciferase expression, correlated with Twist1 expression in vitro, and affected Gata3 and Oct1 binding, these variants may explain part of the observed differences in skin tumor susceptibility at Skts5 between NIH/Ola-S and SPRET/Out-R.

Allele-specific imbalance mapping at human orthologs of mouse susceptibility to colon cancer (Scc) loci.

Colorectal cancer (CRC) can be classified into different types. Chromosomal instable (CIN) colon cancers are thought to be the most common type of colon cancer. The risk of developing a CIN-related CRC is due in part to inherited risk factors. Genome-wide association studies have yielded over 40 single nucleotide polymorphisms (SNPs) associated with CRC risk, but these only account for a subset of risk alleles. Some of this missing heritability may be due to gene-gene interactions. We developed a strategy to identify interacting candidate genes/loci for CRC risk that utilizes both linkage and RNA-seq data from mouse models in combination with allele-specific imbalance (ASI) studies in human tumors. We applied our strategy to three previously identified CRC susceptibility loci in the mouse that show evidence of genetic interaction: Scc4, Scc5 and Scc13. 525 SNPs from genes showing differential expression in the mouse and/or a previous role in cancer from the literature were evaluated for allele-specific imbalance in 194 paired human normal/tumor DNAs from CIN-related CRCs. One hundred three SNPs showing suggestive evidence of ASI (31 variants with uncorrected p values < 0.05) were genotyped in a validation set of 296 paired DNAs. Two variants in SNX10 (SCC13) showed significant evidence of allelic selection after multiple comparisons testing. Future studies will evaluate the role of these variants in combination with interacting genetic partners in colon cancer risk in mouse and humans.

Elevated Mirc1/Mir17-92 cluster expression negatively regulates autophagy and CFTR (cystic fibrosis transmembrane conductance regulator) function in CF macrophages.

Cystic fibrosis (CF) is a fatal, genetic disorder that critically affects the lungs and is directly caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in defective CFTR function. Macroautophagy/autophagy is a highly regulated biological process that provides energy during periods of stress and starvation. Autophagy clears pathogens and dysfunctional protein aggregates within macrophages. However, this process is impaired in CF patients and CF mice, as their macrophages exhibit limited autophagy activity. The study of microRNAs (Mirs), and other noncoding RNAs, continues to offer new therapeutic targets. The objective of this study was to elucidate the role of Mirs in dysregulated autophagy-related genes in CF macrophages, and then target them to restore this host-defense function and improve CFTR channel function. We identified the Mirc1/Mir17-92 cluster as a potential negative regulator of autophagy as CF macrophages exhibit decreased autophagy protein expression and increased cluster expression when compared to wild-type (WT) counterparts. The absence or reduced expression of the cluster increases autophagy protein expression, suggesting the canonical inverse relationship between Mirc1/Mir17-92 and autophagy gene expression. An in silico study for targets of Mirs that comprise the cluster suggested that the majority of the Mirs target autophagy mRNAs. Those targets were validated by luciferase assays. Notably, the ability of macrophages expressing mutant F508del CFTR to transport halide through their membranes is compromised and can be restored by downregulation of these inherently elevated Mirs, via restoration of autophagy. In vivo, downregulation of Mir17 and Mir20a partially restored autophagy expression and hence improved the clearance of Burkholderia cenocepacia. Thus, these data advance our understanding of mechanisms underlying the pathobiology of CF and provide a new therapeutic platform for restoring CFTR function and autophagy in patients with CF.

Evaluation of allele-specific somatic changes of genome-wide association study susceptibility alleles in human colorectal cancers.

BACKGROUND: Tumors frequently exhibit loss of tumor suppressor genes or allelic gains of activated oncogenes. A significant proportion of cancer susceptibility loci in the mouse show somatic losses or gains consistent with the presence of a tumor susceptibility or resistance allele. Thus, allele-specific somatic gains or losses at loci may demarcate the presence of resistance or susceptibility alleles. The goal of this study was to determine if previously mapped susceptibility loci for colorectal cancer show evidence of allele-specific somatic events in colon tumors. METHODS: We performed quantitative genotyping of 16 single nucleotide polymorphisms (SNPs) showing statistically significant association with colorectal cancer in published genome-wide association studies (GWAS). We genotyped 194 paired normal and colorectal tumor DNA samples and 296 paired validation samples to investigate these SNPs for allele-specific somatic gains and losses. We combined analysis of our data with published data for seven of these SNPs. RESULTS: No statistically significant evidence for allele-specific somatic selection was observed for the tested polymorphisms in the discovery set. The rs6983267 variant, which has shown preferential loss of the non-risk T allele and relative gain of the risk G allele in previous studies, favored relative gain of the G allele in the combined discovery and validation samples (corrected p-value = 0.03). When we combined our data with published allele-specific imbalance data for this SNP, the G allele of rs6983267 showed statistically significant evidence of relative retention (p-value = 2.06×10(-4)). CONCLUSIONS: Our results suggest that the majority of variants identified as colon cancer susceptibility alleles through GWAS do not exhibit somatic allele-specific imbalance in colon tumors. Our data confirm previously published results showing allele-specific imbalance for rs6983267. These results indicate that allele-specific imbalance of cancer susceptibility alleles may not be a common phenomenon in colon cancer.