A Strategy to Identify Dominant Point Mutant Modifiers of a Quantitative Trait

Abstract

A central goal in the analysis of complex traits is to identify genes that modify a phenotype. Modifiers of a cancer phenotype may act either intrinsically or extrinsically on the salient cell lineage. Germline point mutagenesis by ethylnitrosourea can provide alleles for a gene of interest that include loss-, gain-, or alteration-of-function. Unlike strain polymorphisms, point mutations with heterozygous quantitative phenotypes are detectable in both essential and nonessential genes and are unlinked from other variants that might confound their identification and analysis. This report analyzes strategies seeking quantitative mutational modifiers of ApcMin in the mouse. To identify a quantitative modifier of a phenotype of interest, a cluster of test progeny is needed. The cluster size can be increased as necessary for statistical significance if the founder is a male whose sperm is cryopreserved. A second critical element in this identification is a mapping panel free of polymorphic modifiers of the phenotype, to enable low-resolution mapping followed by targeted resequencing to identify the causative mutation. Here, we describe the development of a panel of six “isogenic mapping partner lines” for C57BL/6J, carrying single-nucleotide markers introduced by mutagenesis. One such derivative, B6.SNVg, shown to be phenotypically neutral in combination with ApcMin, is an appropriate mapping partner to locate induced mutant modifiers of the ApcMin phenotype. The evolved strategy can complement four current major initiatives in the genetic analysis of complex systems: the Genome-wide Association Study; the Collaborative Cross; the Knockout Mouse Project; and The Cancer Genome Atlas.

The utility of Apc-mutant rats in modeling human colon cancer

“…The strong effects of genetic background on the Pirc phenotype can be dissected by the methods of quantitative trait genetics. Thanks to the efficient ENU mutagenesis of the rat germline cited above, the genetic system impacting the biology of the Pirc strain can be extended by mutagenesis beyond the viable polymorphic alleles present in inbred strains ( Dove et al, 2014 ). Compared with the Modifier-of-Min loci of the mouse, Pirc modifiers affecting colonic tumors are more likely to be orthologous to such loci discovered by genome-wide association analyses in human populations ( Dunlop et al, 2012 ; Peters et al, 2013 ).…”

The conserved protective cyclic AMP-phosphodiesterase function PDE4B is expressed in the adenoma and adjacent normal colonic epithelium of mammals and silenced in colorectal cancer

“…This study and that of the gender effect [ 3 ] show that these two regions of the intestinal tract differ in their response to genetic modifiers. How the understanding of the complexities of colon cancer being unraveled by mutational [ 62 ] and phenotypic [ 63 ] analysis can be conceptually simplified into metaphors including driver, landscaper and growth rate regulator [ 33 , 64 ] is unclear. It seems possible that an indefinitely large number of functions can impact colonic neoplasia; such an “Infinite Tree” can be explored by the expansion of modifier genetics [ 62 ] and conservation of molecular signals across multiple genera.…”

“…A 12:12 hour light:dark cycle was maintained throughout the experiments. C57BL/6J Apc Min /+ mice (developed in the laboratory of WFD and commercially available through the Jackson Laboratory, Bar Harbor, ME) were maintained as a closed colony, C57BL/6JD [ 62 ] by breeding Apc +/+ females to Apc Min /+ males and monitoring the canonical high tumor number [ 1 ] (University of Missouri Stock 043849-MU). F 1 generation (C57BL/6JD x BTBR)F 1 -Min mice were generated by breeding female C57BL/6JD Apc Min/+ to male BTBR mice.…”