Single Nucleotide Polymorphisms within Interferon Signaling Pathway Genes Are Associated with Colorectal Cancer Susceptibility and Survival

<div><p>Interferon (IFN) signaling has been suggested to play an important role in colorectal carcinogenesis. Our study aimed to examine potentially functional genetic variants in interferon regulatory factor 3 (<i>IRF3</i>), <i>IRF5</i>, <i>IRF7</i>, type I and type II <i>IFN</i> and their receptor genes with respect to colorectal cancer (CRC) risk and clinical outcome. Altogether 74 single nucleotide polymorphisms (SNPs) were covered by the 34 SNPs genotyped in a hospital-based case-control study of 1327 CRC cases and 758 healthy controls from the Czech Republic. We also analyzed these SNPs in relation to overall survival and event-free survival in a subgroup of 483 patients. Seven SNPs in <i>IFNA1</i>, <i>IFNA13</i>, <i>IFNA21</i>, <i>IFNK</i>, <i>IFNAR1</i> and <i>IFNGR1</i> were associated with CRC risk. After multiple testing correction, the associations with the SNPs rs2856968 (<i>IFNAR1</i>) and rs2234711 (<i>IFNGR1</i>) remained formally significant (<i>P</i> = 0.0015 and <i>P</i><0.0001, respectively). Multivariable survival analyses showed that the SNP rs6475526 (<i>IFNA7/IFNA14</i>) was associated with overall survival of the patients (<i>P</i> = 0.041 and event-free survival among patients without distant metastasis at the time of diagnosis, <i>P</i> = 0.034). The hazard ratios (HRs) for rs6475526 remained statistically significant even after adjustment for age, gender, grade and stage (<i>P</i> = 0.029 and <i>P</i> = 0.036, respectively), suggesting that rs6475526 is an independent prognostic marker for CRC. Our data suggest that genetic variation in the IFN signaling pathway genes may play a role in the etiology and survival of CRC and further studies are warranted.</p></div

Associations between candidate SNPs and colorectal cancer susceptibility.

1<p>Number of cases may differ due to missing data.</p>2<p>Two-sided X² test for genotype distribution between the cases and controls, adjusted for age and gender.</p><p>No., number of subjects; OR, odds ratio; CI, confidence interval. Bold numbers indicate a statistical significance at 5% level.</p><p>Bold numbers in Italics indicate a statistical significance at 5% level after adjustment for multiple comparisons.</p><p>Associations between candidate SNPs and colorectal cancer susceptibility.</p

Association of rs7047687, rs6745526 and rs11770589 with overall and event-free survival of newly diagnosed colorectal cancer patients.

1<p>Overall survival was calculated for all patients diagnosed between 2003 and 2010 (n = 483).</p>2<p>Event-free survival was calculated for patients diagnosed between 2003 and 2010, who did not have distant metastasis at the time of diagnosis</p><p>(n = 325).</p>3<p>Number of cases may differ due to missing data.</p><p>No., number of patients; OR, odds ratio; CI, confidence interval; M = 0, no distant metastasis present Bold numbers indicate a statistical significance at 5% level.</p><p>Association of rs7047687, rs6745526 and rs11770589 with overall and event-free survival of newly diagnosed colorectal cancer patients.</p

Characteristics of the 483 newly diagnosed Czech colorectal cancer patients.

<p>No., number of patients; T, size or direct extent of the primary tumor; N, degree of spread to regional lymph nodes; M, presence of metastasis.</p><p>Characteristics of the 483 newly diagnosed Czech colorectal cancer patients.</p

Polymorphisms evaluated in this study.

1<p>Minor allele frequency (MAF) based on Utah residents with Northern and Western European ancestry from the CEPH collection in the HapMap project.</p>2<p>Pairwise linkage disequilibrium (r²) was calculated for the SNPs with MAF≥10% within the regions of interest based on Utah residents with Northern and Western European ancestry from the CEPH collection in the HapMap project.</p>3<p>Because no assays were available for the potentially functionally SNPs, the SNPs rs6475526, rs1874327 and rs2856968, respectively, were genotyped instead.</p><p>Polymorphisms evaluated in this study.</p

Kaplan-Meier analysis of survival according to genotypes of SNPs rs6475526, located 5′ to IFNA14, and capturing two IFN7 promoter SNPs and rs7047687 located in IFNA21 promoter.

<p>(A) Overall survival among all colorectal cancer patients (rs6475526, n = 465). (B) Event-free survival among patients without distant metastasis at diagnosis (rs6477526, n = 310). (C) Overall survival among all colorectal cancer patients (rs7047687, n = 464).</p

Sequence diversity of kappa light chains from patients with AL amyloidosis and multiple myeloma

AL amyloidosis (AL) results from the misfolding of immunoglobulin light chains (IG LCs). Aim of this study was to comprehensively analyse kappa LC sequences from AL patients in comparison with multiple myeloma (MM). We analysed IGKV/IGKJ usage and associated organ tropism and IGKV1/D-33 in terms of mutational analysis and theoretical biochemical properties. cDNA and bulk RNA sequencing of the LCs of AL and MM patients. We studied 41 AL and 83 MM patients showing that IGKV1 was most expressed among kappa AL and MM, with higher frequency in AL (80% vs. 53%, p = .002). IGKV3 was underrepresented in AL (10% vs. 30%, p = .014). IGKJ2 was more commonly used in AL than in MM (39% vs. 29%). Patients with IGKV1/D-33 were associated with heart involvement (75%, p = .024). IGKV1/D-33-segments of AL had a higher mutation count (AL = 12.0 vs. MM = 10.0). FR3 and CDR3 were most frequently mutated in both, with a median mutation count in FR3 being the highest (AL = 4.0; MM = 3.5) and one mutation hotspot (FR3 (83I)) for IGKV1/D-33/IGKJ2 was associated with cardiac involvement. This study confirmed that germline usage has an influence on AL amyloidosis risk and organ involvement.</p

CRC risk: genotype distribution of SNPs analyzed in the Czech case-control population for SNPs with p ≤ 0.05.

<p>Amino acid changes are given as <> with the amino acid position indicated. Data adjusted for age at diagnosis and sex. Nominal significance at p≤0.05; significance level corrected for multiple testing (39 genotyped SNPs) at p≤0.001.</p

Expression of selected CRC-associated NLRs in immune cells, primary tissue samples or CRC cell lines.

<p>mRNA expression of NLRP2 (A,B), NLRP5 (C), NLRP13 (D), NLRC5 (E) and NLRP3 (F) was determined relative to the housekeeper TBP by performing triplicate (means +SD show) qPCR using TaqMan gene-specific primers and probes on the indicated samples (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199350#sec002" target="_blank">Methods</a>). In the case of (E) HCT cells were treated with 1000 U/ml IFNγ or 50 ng/ml S. typhimurium Flagellin for 3 or 6 hours as indicated. TBP-relative ΔCt values were normalized to a reference sample (labelled R, ΔΔCt method). N denotes samples in which no expression was detectable above Ct within 40 cycles.</p