Evaluation of Allele-Specific Somatic Changes of Genome-Wide Association Study Susceptibility Alleles in Human Colorectal Cancers

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.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.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)).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

CRC risk-associated GWAS SNPs assessed for allele-specific imbalance in the present study.

*<p>Position by UCSC Genome Browser (Human Feb. 2009 GRCh37/hg19 Assembly).</p>†<p>MAF = Minor Allele Frequency, as listed on NCBI dbSNP.</p>§<p>Odds Ratio from first listed GWAS reference (95% confidence interval). NA = not available.</p>‡<p>P-value from first listed GWAS reference.</p>∞<p>SNP positions in modest linkage disequilibrium (D′ = 0.76, ref. 15).</p>#<p>SNP positions in modest linkage disequilibrium (D′ = 0.71, ref. 15).</p>▵<p>SNP positions in high linkage disequilibrium (D′ = 0.99, ref. 9).</p

Analysis of allele-specific imbalance in discovery sample set.

*<p><i>Risk Allele Lost</i> refers to relative loss of the risk allele compared to the non-risk allele. Number in parentheses indicates percentage of total heterozygous samples showing relative loss of risk allele.</p>†<p><i>Non-risk Allele Lost</i> refers to relative loss of the non-risk allele compared to the risk allele. Number in parentheses indicates percentage of total heterozygous samples showing relative loss of non-risk allele.</p>§<p>Total number of tumors with imbalance/total heterozygous samples (% of heterozygotes showing imbalance).</p>‡<p>Chi-squared statistical test, df = 1. Unadjusted for multiple comparisons.</p

Analysis of allele-specific imbalance in discovery, validation, and combined sample sets.

*<p><i>Risk Allele Lost</i> refers to relative loss of risk allele compared to non-risk allele. Number in parentheses indicates percentage of total heterozygous samples showing relative loss of risk allele.</p>†<p><i>Non-risk Allele Lost</i> refers to relative loss of non-risk allele compared to risk allele. Number in parentheses indicates percentage of total heterozygous samples showing relative loss of non-risk allele.</p>§<p>Total number of tumors with imbalance/total heterozygous samples (% of heterozygotes showing imbalance).</p>‡<p>Chi-squared statistical test, df = 1.</p>∞<p>Bonferroni correction for 16 multiple comparisons (original) or 3 multiple comparisons (validation, combined).</p

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

<p>Cystic fibrosis (CF) is a fatal, genetic disorder that critically affects the lungs and is directly caused by mutations in the <i>CF transmembrane conductance regulator (CFTR)</i> 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 (<i>Mirs</i>), and other noncoding RNAs, continues to offer new therapeutic targets. The objective of this study was to elucidate the role of <i>Mirs</i> 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 <i>Mirc1/Mir17-92</i> 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 <i>Mirc1/Mir17-92</i> and autophagy gene expression. An <i>in silico</i> study for targets of <i>Mirs</i> that comprise the cluster suggested that the majority of the <i>Mirs</i> 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 <i>Mirs</i>, via restoration of autophagy. In vivo, downregulation of <i>Mir17</i> and <i>Mir20a</i> partially restored autophagy expression and hence improved the clearance of <i>Burkholderia cenocepacia</i>. 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.</p

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