Mouse Genome-Wide Association Mapping Needs Linkage Analysis to Avoid False-Positive Loci

We carried out genome-wide association (GWA) studies in inbred mouse strains characterized for their lung tumor susceptibility phenotypes (spontaneous or urethane-induced) with panels of 12,959 (13K) or 138,793 (140K) single-nucleotide polymorphisms (SNPs). Above the statistical thresholds, we detected only SNP rs3681853 on Chromosome 5, two SNPs in the pulmonary adenoma susceptibility 1 (Pas1) locus, and SNP rs4174648 on Chromosome 16 for spontaneous tumor incidence, urethane-induced tumor incidence, and urethane-induced tumor multiplicity, respectively, with the 13K SNP panel, but only the Pas1 locus with the 140K SNP panel. Haplotype analysis carried out in the latter panel detected four additional loci. Loci reported in previous GWA studies failed to replicate. Genome-wide genetic linkage analysis in urethane-treated (BALB/c×C3H/He)F2, (BALB/c×SWR/J)F2, and (A/J×C3H/He)F2 mice showed that Pas1, but none of the other loci detected previously or herein by GWA, had a significant effect. The Lasc1 gene, identified by GWA as a functional element (Nat. Genet., 38:888–95, 2006), showed no genetic effects in the two independent intercross mouse populations containing both alleles, nor was it expressed in mouse normal lung or lung tumors. Our results indicate that GWA studies in mouse inbred strains can suffer a high rate of false-positive results and that such an approach should be used in conjunction with classical linkage mapping in genetic crosses

Interferon signaling in viral hepatitis

Hepatitis C virus (HCV) is a single stranded positive RNA virus classified in 6 different genotypes. Hepatocytes are the main targets of HCV infection. It has been estimated that 60 to 70% of the infected patients develop chronic infection. If left untreated, chronic hepatitis C (CHC) results in cirrhosis in 10 to 20% of the cases. Once cirrhosis is established, the risk of hepatocellular carcinoma (HCC) development increases dramatically, with an estimated annual rate of 1% to 4%. The standard of care (SOC) for CHC treatment is based on pegylated IFN? (peg-IFN?) and Ribavirin administration. Peg-IFN? injection activates the Jak-STAT signaling pathway that leads to the phosphorylation of STAT1 and culminates in the up-regulation of hundred of genes in the liver, establishing an antiviral state. However, peg-IFN?-based therapy achieves the clearance of HCV only in half of the chronic infected individuals. In the recent past, the lack of response to peg-IFN?-based therapy in CHC have been associated to the broad up-regulation of interferon regulated genes (IRGs) in the liver of CHC patients, already before treatment. The reason why the pre-activated hepatic IFN system fails to clear HCV remains to be elucidated. Furthermore, the molecular mechanisms that define the level of activation of the hepatic IFN system in CHC are not clear. In the recent past, several genome-wide association studies have reported a strong association of treatment-failure with minor (less frequent in the population) alleles at single nucleotide polymorphisms (SNPs) located in the IL28B locus on chromosome 19. Minor alleles at SNPs in the IL28B locus have also been associated to the up-regulation of the hepatic IFN system pre-treatment in CHC patients. So far the molecular mechanisms that links allelic variants at IL28B locus, the pre-activation of the hepatic IFN system and treatment-response in CHC patients remain to be elucidated. The present work is aimed to investigate two of the possible molecular mechanisms that could mediate the pre-activation of the IFN system in the liver of CHC patients that do not respond to therapy. In the first part of the thesis the role of unphosphorylated-STAT1 (U-STAT1) in mediating the up-regulation of hepatic IRGs in CHC patients was investigated. We have reported that STAT1 accumulates in the liver of CHC patients non-responders. Furthermore, experimental evidences suggest that STAT1 could play a role as transcription factor independently by its phosphorylation on tyrosine 701 and its unphosphorylated form can drive the expression of a subset of IRGs. In the present study we took advantage of a cell line constitutively lacking STAT1 expression and we exogenously re-expressed a mutant form of STAT1 that can not be phosphorylated, mimicking U-STAT1. We proved that U-STAT1 per se is not able to induce the expression of IRGs and it is unlikely to be the cause of the pre-activated IFN system observed in the liver of non-responders CHC patients. In the second part of the thesis, we investigated the role of IFN?s signaling pathway in the definition of the pre-activated hepatic IFN system in CHC. IFN?s are the most recently group of IFNs. IFN?s signal through the cells via a different receptor compared to the one of IFN?. However, the intracellular signaling pathway of the two class of cytokines is completely overlapping, leading to the up-regulation of the same IRGs. We demonstrated that in an hepatoma cell line Huh7 the over-expression of IL28R?, one of the two chains of INF? receptor complex, mediates the long lasting up-regulation of IRGs upon IFN? stimulation. We confirmed our results in human liver biopsies, where we found a significant positive correlation between IL28R? and IRGs expression. We observed that IL28R? is an IRG itself but its level of expression is modulated by allelic variants at SNPs mapping in the IL28B locus, that have been associated to treatment response in CHC patients. In conclusion we provide evidences of a molecular mechanism that links the pre-activation of the hepatic IFN system (and non-response) and allelic variants at IL28B locus

Mouse Pulmonary Adenoma Susceptibility 1 Locus Is an Expression QTL Modulating Kras-4A

Pulmonary adenoma susceptibility 1 (Pas1) is the major locus responsible for lung tumor susceptibility in mice; among the six genes mapping in this locus, Kras is considered the best candidate for Pas1 function although how it determines tumor susceptibility remains unknown. In an (A/J×C57BL/6)F4 intercross population treated with urethane to induce lung tumors, Pas1 not only modulated tumor susceptibility (LOD score = 48, 69% of phenotypic variance explained) but also acted, in lung tumor tissue, as an expression quantitative trait locus (QTL) for Kras-4A, one of two alternatively spliced Kras transcripts, but not Kras-4B. Additionally, Kras-4A showed differential allelic expression in lung tumor tissue of (A/J×C57BL/6)F4 heterozygous mice, with significantly higher expression from the A/J-derived allele; these results suggest that cis-acting elements control Kras-4A expression. In normal lung tissue from untreated mice of the same cross, Kras-4A levels were also highly linked to the Pas1 locus (LOD score = 23.2, 62% of phenotypic variance explained) and preferentially generated from the A/J-derived allele, indicating that Pas1 is an expression QTL in normal lung tissue as well. Overall, the present findings shed new light on the genetic mechanism by which Pas1 modulates the susceptibility to lung tumorigenesis, through the fine control of Kras isoform levels. © 2014 Dassano et al

IFN-λ receptor 1 expression is induced in chronic hepatitis C and correlates with the IFN-λ3 genotype and with nonresponsiveness to IFN-α therapies

The molecular mechanisms that link IFN-λ3 genotypes to differential induction of interferon (IFN)-stimulated genes (ISGs) in the liver of patients with chronic hepatitis C (CHC) are not known. We measured the expression of IFN-λ and of the specific IFN-λ receptor chain (IFN-λR1) in 122 liver biopsies of patients with CHC and 53 control samples. The IFN-λ3 genotype was not associated with differential expression of IFN-λ, but rather IFN-λR1. In a series of 30 primary human hepatocyte (PHH) samples, IFN-λR1 expression was low but could be induced with IFN-α. IFN-α-induced IFN-λR1 expression was significantly stronger in PHHs carrying the minor IFN-λ3 allele. The analysis of liver biopsies of patients with CHC revealed a strong association of high IFN-λR1 expression with elevated ISG expression, with IFN-λ3 minor alleles, and with nonresponse to pegylated IFN-α and ribavirin. The findings provide a missing link between the IFN-λ3 genotype and the associated phenotype of treatment nonresponse

Genome-wide genetic linkage analysis of loci affecting urethane-induced lung tumor multiplicity.

<p>(A) (BALB/c×C3H/He)F2 cross detects the <i>Pas1</i> locus at LOD score = 18.4. (B) (A/J×C3H/He)F2 cross detected the <i>Pas1</i> locus at LOD score = 18.7. Red curves indicate the results of the composite interval mapping, whereas black curves indicate the results of genome scan using the <i>Kras</i> genotype as covariate (conditioning on the <i>Pas1</i> alleles). Horizontal lines indicate the threshold values (α = 0.05) of the LOD score. The <i>Clas2</i> locus (Chromosome 4) showed no significant linkage, despite the presence of the claimed functional polymorphism (D102E) in both crosses. No other locus detected by whole-genome strain survey showed significant linkage.</p

Spontaneous lung tumor incidence correlates with both urethane-induced lung tumor multiplicity (green) and incidence (red) in mouse inbred strains.

<p>Incidence is given as mean percentages, whereas multiplicity is mean number of tumors/mouse. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000331#pgen-1000331-t001" target="_blank">Table 1</a> for phenotype values.</p

Threshold probabilities (expressed as −log P) corresponding to experiment-wide type I risk errors α = 0.05 and α = 0.10.

<p>Threshold probabilities (expressed as −log P) corresponding to experiment-wide type I risk errors α = 0.05 and α = 0.10.</p

Haplotype-associated lung tumor modifier (<i>Halt</i>) loci identified by haplotype analysis, using the 140K BROAD SNP panel.

a<p>Position of the central SNP, in mega bases (Mb) based on NCBI m37 mouse assembly.</p>b<p>Global -log P is the minus logarithm of the p-value for the haplotype sliding window (window size: 3-SNPs).</p>c<p>UI, urethane-induced lung tumor incidence, UM, urethane-induced lung tumor multiplicity.</p>d<p>At each <i>Halt</i> locus, the intercrosses whose parental strains carry different alleles, and that have herein been analyzed, are indicated. AHF2, (A/J×C3H/He)F2; CHF2, (BALB/c×C3H/He)F2; CWF2, (BALB/c×SWR/J)F2.</p