The rate-limiting step in ENU mutagenesis has long been the identification of causative mutations. Here we report a cost-effective approach to identify phenotype-related nucleotide variants. Specifically, we targeted the Lampe1 locus—an ENU germline mutant exhibiting growth retardation and spontaneous hepatosteatosis, evolving to steatohepatitis and hepatocellular carcinoma. We performed low resolution mapping based on a limited number of meioses resulting in the identification of a ~14 Mb critical region. Subsequently we applied targeted exon enrichment—with the inclusion of 50 bp proximal/distal flanking sequence for all exons—and analyzed the enriched DNA using next-generation sequencing technology. The approach effectively identified a donor splice site mutation in the Palmitoyl Acetyl-coenzyme A oxygenase-1 (Acox1) gene that was responsible for the described phenotype in Lampe1 mice. Importantly, this approach eliminated the necessity for fine-mapping—a laborious, time consuming and relative expensive process related to forward genetics and ENU mutagenesis. The coverage of targeted DNA for the Lampe1 critical region was exceptional, with 98.1% of the targeted region being sequenced with a minimal depth of 10. This exceptionally high coverage was predominantly due to the fact that a relatively small genomic interval was targeted (690.7 kb of targeted DNA). The Nimblegen array used in this study has 384,000 probes and a capacity to capture up to 5 Mb of DNA sequence. This means that the tiling across the target region was approximately 1 probe every 1 or 2 bases on average. This optimal capture design, combined with the large number of reads generated (average coverage depth of 453× over the target region) were critical elements that allowed us to obtain 10× coverage on 98.1% of the target region.
Although the size and gene density of a critical region may vary somewhat between ENU germline mutants, we anticipate that the obtained sequence coverage can generally be reproduced with a similar probe design and total number of reads generated. In addition, the enrichment capacity of 2.1 M arrays captures ~30 Mb of targeted sequence, allowing for the analysis of the entire mouse exome. Nonetheless, causality (i.e. through linkage analysis or genetic confirmation) will remain an essential aspect of forward genetics.
The effective identification of genomic nucleotide changes also expands the possibilities in (immune)phenotyping. In many instances, phenotypes are lost or significantly influenced by modifier loci located on outcross strains carrying a high degree of genetic variation. For example, the genetic dissection of NK cell function has proven challenging in our laboratory because of the large variation in NK cell ligands/receptors existing on different mouse backgrounds (Hoebe, unpublished results). The current approach avoids fine mapping and allows for the exploration of phenotypes that are more subtle and can be traced following the outcross to strains with minimal genetic variation (e.g. the C57BL/6J and C57BL/10J strains). Thus the ability to effectively target large genomic intervals not only facilitates mutation discovery, but also expands the number of phenotypes that can be explored. Interestingly, ENU has been described as a powerful mutagen that at optimal doses can induce about one base-pair change per million base-pairs of genomic DNA 
. Here we identified and confirmed 3 base-pair changes in 690 kb of sequenced DNA, suggesting a higher mutation frequency induced by ENU than previously estimated. Further analysis of large areas of genomic DNA using next-generation sequencing should ultimately provide a better idea of the exact mutation frequency for ENU in mice.
The pathogenesis observed in Acox1lampe1
mice largely resembles the pathogenesis observed in Acox1
null mice as previously published by Reddy's group 
. The latter presented growth retardation, severe microvesicular hepatic steatosis and sustained activation of peroxisome proliferator-activated receptor-α (PPARα) 
. Acox1 is expressed as a 72 kDa precursor protein that enters the peroxisome via the C-terminal Ser-Lys-Leu-related tripeptide target signal 
. Within the peroxisome, Acox1 is cleaved by the protease trypsin domain-containing 1 (Tysdn1), resulting into a 50 kDa N-terminal and a 22 kDa C-terminal subunit ultimately generating heterodimeric complexes comprised of all three subunits 
. The Lampe1
mutation affects the C-terminal domain of Acox1 containing the Acetyl-CoA oxidase domain—an essential domain for enzyme activity. The 48 amino acid deletion, however, does not affect the C-terminal signal peptide (SKL) nor does it affect the Tysdn1-specific cleavage site, suggesting normal localization and processing of the mutant Acox1 protein in the peroxisome may still occur. Indeed, immunoblot analysis reveals a quantitative similar expression of Acox1, albeit with a reduced molecular weight in liver and kidney of Acox1lampe1
mice. Although we have not performed enzymatic activity assays to confirm the loss of Acox1 function in Lampe1 mutant mice, the 48 amino-acid deletion from the Acetyl-CoA oxidase domain is likely to render the protein functionally deficient and cause a phenotype similar to the previously reported Acox1
null mice. Nonetheless, subtle differences seem apparent between both mouse models. First of all, the distribution of microvesicular fatty metamorphosis in 2-month-old Acox1lampe1
livers is zonal and observed in zones 2 and 3 but not zone 1, whereas Fan et al reported fatty changes in all hepatocytes irrespective of their distribution in the liver of Acox1−/−
mice at similar age 
. Notably, we observed a more severe and uniform fatty metamorphosis in the liver following the outcross of the Lampe1
mutation onto 129X1/SvJ background (results not shown), suggesting that modifier loci on the 129X1/SvJ or C57BL/6J to some extent influence the degree of hepatosteatosis.
Acox1 is essential for β-oxidation of very-long chain fatty acyl-CoA and arachidonic acid metabolites such as 8(S)-hydroxy-eicosatetraenoic acid and leukotriene B4
). In the absence of functional Acox1, these lipid metabolites accumulate and act as natural ligands for the nuclear receptor PPARα 
, initiating a unique transcriptional program following heterodimeric association of PPARα with nuclear retinoid X receptor (RXR) 
. Ultimately, this complex induces a select set of genes encoding the peroxisomal β-oxidation system, including Acox1 but also enzymes such as cytochrome P450 4A isoforms (CYP4A1 and CYP4A3) 
. The latter metabolize long-chain fatty acids via the ω-oxidation pathway. In the absence of functional Acox1, this pathway becomes an important metabolic pathway for long-chain fatty acids, resulting in accumulation of toxic dicarboxilic acids (DCAs) causing mitochondrial damage 
. In addition, the dramatic increase in enzyme expression related to lipid oxidation coincides with high levels of reactive oxygen species such as hydrogen peroxide, ultimately causing cell death, regenerative cell proliferation and hepatocarcinogenesis 
. Indeed, compound homozygous PPARα−/−
mice lack increased peroxisomal proliferation and exhibit reduced steatosis and liver cell proliferation 
, thus verifying the essential role of PPARα in the pathogenesis of Acox1
-deficient mice. Although in our Acox1lampe1
model we have not directly measured the activation of PPARα and expression of CYP450 enzymes, the morphologic changes in the liver of Acox1lampe1
mice were similar to the previously published Acox1
-null mice by Reddy's group. Finally, the increased number of peroxisomes in hepatocytes was a common feature in both Acox1−/−
mice. In addition, stress changes in the mitochondria were prevalent in Acox1lampe1
Recent epidemiological reports suggest that obesity significantly increases the risk of hepatocellular carcinoma in humans 
. Obesity is associated with simple steatosis (60%), nonalcoholic steatohepatitis (25%) whereas 3% to 5% of obese people exhibit liver cirrhosis 
. Steatosis is associated with increased levels of ROS and the induction of low level inflammatory response including the release of proinflammatory cytokines such TNFα 
. A report by Park et al suggests that low-level inflammation, specifically the activation of STAT3 via cytokines such as IL-6, is a requirement for development of hepatocellular carcinoma in mouse models with dietary or genetic obesity 
. Currently, the molecular basis for (sterile) low level inflammation in Acox1-deficient mice or for that matter in the context of obesity is poorly defined. An important trigger of inflammation may be derived from the excessive cell death—possibly due to CYP4A-mediated ROS production and DCA-release—observed in Acox1
-deficient mice. This may induce further release of lipids and other components sensed by innate immune receptors ultimately causing steatohepatitis, cell proliferation and hepatocellular carcinoma. A large number of studies have reported the induction of proinflammatory cytokines following exposure of dendritic cells or macrophages to cell death 
. The regulatory pathways and innate receptors involved in mediating inflammation via cell death have been poorly defined particularly in the context of dying cells with excessive lipid accumulation as observed in Acox1
-deficient mice. Thus Acox1lampe1
mice may present a unique model to further dissect the (sterile) inflammatory pathways that drive liver regeneration and ultimately hepatocellular carcinoma.