Identification of five novel modifier loci of ApcMin harbored in the BXH14 recombinant inbred strain
Advance Access Publication May
Identification of five novel modifier loci of ApcMin harbored in the BXH14 recombinant inbred strain
Stephanie C.Nnadi 1
Rayneisha Watson 1
Julie Innocent 1
Gregory E.Gonye 0 1
Arthur M.Buchberg 1
Linda D.Siracusa 1
0 Department of Pathology, Anatomy, and Cell Biology, Kimmel Cancer Center, Thomas Jefferson University , Philadelphia, PA 19107-5541 , USA
1 Department of Microbiology and Immunology
Every year thousands of people in the USA are diagnosed with small intestine and colorectal cancers (CRC). Although environmental factors affect disease etiology, uncovering underlying genetic factors is imperative for risk assessment and developing preventative therapies. Familial adenomatous polyposis is a heritable genetic disorder in which individuals carry germ-line mutations in the adenomatous polyposis coli (APC) gene that predisposes them to CRC. The ApcMin mouse model carries a point mutation in the Apc gene and develops polyps along the intestinal tract. Inbred strain background influences polyp phenotypes in ApcMin mice. Several Modifier of Min (Mom) loci that alter tumor phenotypes associated with the ApcMin mutation have been identified to date. We screened BXH recombinant inbred (RI) strains by crossing BXH RI females with C57BL/6J (B6) ApcMin males and quantitating tumor phenotypes in backcross progeny. We found that the BXH14 RI strain harbors five modifier loci that decrease polyp multiplicity. Furthermore, we show that resistance is determined by varying combinations of these modifier loci. Gene interaction network analysis shows that there are multiple networks with proven gene-gene interactions, which contain genes from all five modifier loci. We discuss the implications of this result for studies that define susceptibility loci, namely that multiple networks may be acting concurrently to alter tumor phenotypes. Thus, the significance of this work resides not only with the modifier loci we identified but also with the combinations of loci needed to get maximal protection against polyposis and the impact of this finding on human disease studies.
Colorectal cancer (CRC) is the third most deadly cancer among men
and women in the USA (www.cancer.org). Familial adenomatous
polyposis (FAP) is an autosomal dominant genetic disease
characterized by the development of hundreds to thousands of adenomas in the
colon beginning in the second decade of life (
). Patients affected by
FAP carry a mutation in the adenomatous polyposis coli (APC) gene
). These individuals have an ~100% chance of developing CRC by
the age of 40 years (
Mouse models have proven to be powerful tools in the study of
intestinal and CRCs. Several mouse models of FAP have been
generated over the past two decades (www.informatics.jax.org). The ApcMin
model was generated through an ethylnitrosourea mutagenesis screen
). The ApcMin allele has a T-to-A transversion that creates a
premature stop at codon 850 (
). Mice that carry the ApcMin/+ mutation
on the C57BL/6J (B6) background develop 50 or more polyps along
the length of the intestinal tract (
). B6 ApcMin mice are moribund by
150 days of age due to severe anemia and polyp-induced intestinal
Abbreviations: APC, adenomatous polyposis coli; GWAS, genome-wide
association studies; QTL, quantitative trait loci; SNP, single-nucleotide
Inbred strain background is known to influence the ApcMin
phenotype. Hybrid progeny from a cross between B6 ApcMin/+ mice and
AKR/J (AKR), C3H/HeJ (C3H), MA/MyJ, 129P2, or Mus musculus
castaneus (CAST/EiJ) mice show a dramatic decrease in polyp number
as well as an increase in lifespan (
). Conversely, when the ApcMin
mutation is carried on the BTBR genetic background, mice develop
>600 intestinal polyps and become moribund by 60 days of age (16).
The reason for these differences in the ApcMin phenotype between
inbred strains has been attributed to modifier genes (
modifier loci of the ApcMin mutation have already been identified (
In particular, the Modifier of Min 1 (Mom1), located on chromosome
(Chr) 4, lowers polyp number and size in ApcMin mice (
secretory type II non-pancreatic phospholipase A2 (Pla2g2a) gene is
responsible for most of the Mom1 phenotype (
Previously, we showed that the C3H inbred strain harbors
modifier loci of the ApcMin mutation (
). Even without the presence of a
resistant Mom1R allele, offspring from a cross of congenic C3H.B6
Mom1S/S females to B6 ApcMin/+ mice exhibited significantly fewer
polyps than B6 ApcMin/+ controls (
). To further investigate the
modifier loci present in the C3H genome, we chose to evaluate the BXH
recombinant inbred (RI) strains, whose progenitors are B6 and C3H
). RI strains are established by crossing two independent inbred
strains followed by sequential brother-sister mating for 20
generations starting at the F2 generation (
). Each strain of an RI series
has inherited a unique homozygous set of progenitor alleles, with an
average of 50% of their genome contributed from one parental strain
and 50% of their genome contributed from the other parental strain.
Homozygous clustering, in addition to allelic variation, present in
RI strains makes them useful tools for mapping complex traits (
Advantages to using RI strains as opposed to common inbred strains
include (i) identification of problems arising from linkage
disequilibrium within existing sets, (ii) large RI sets can provide greater
statistical power and precision for finer resolution mapping of complex
trait loci, and (iii) large RI sets can be utilized for dissecting factors
that influence susceptibility, predisposition, penetrance, and
expressivity of complex traits (
Recombinant congenic (RC) strains are also powerful resources in
the search for multiple genes involved in quantitative traits (
RC strains are constructed in a similar way to RI strains, starting with
a cross between two parental inbred strains (
). Offspring are then
selected randomly and backcrossed to one of the parental strains for
the next two generations. From this point on, brother-sister mating
proceeds for at least 14 generations, creating mice that carry an
average of 87.5% of their genome from the recipient progenitor strain and
12.5% of their genome from the donor progenitor strain.
The unique RI and RC strains of the BXH series allow for the
identification of modifier loci that were inherited from either the B6 or C3H
progenitor strains. To eliminate the protective effect of the resistant
Mom1R locus (present in the progenitor C3H strain), we selected only
BXH RI and RC strains that were homozygous for the same susceptible
Mom1S locus carried by the progenitor B6 strain. The range of polyp
numbers observed in the RI and RC strains extended from the high to
the low parental control groups. To our knowledge, this is the first time
that a set of RI strains have been evaluated for their impact on the ApcMin
phenotype. In this study, we report the identification of five modifier
loci (named Mom14, Mom15, Mom16, Mom17 and Mom18) that lower
polyp multiplicity in the small intestine and colon.
Materials and methods
The BXH2/TyJ, BXH4/TyJ, BXH8/TyJ, BXH14/TyJ, BXH22/KccJ, and
B6cC3-1/KccJ mice were purchased from The Jackson Laboratory (Bar
Harbor, ME). The BXH22 RI line, the B6cC3-1 RC line, and the C3H.B6
Mom1S/S congenic line were generated by the Siracusa laboratory at the
Kimmel Cancer Center (KCC) (
). The B6cC3-1 RC strain was derived from
an initial intercross between the albino B6 Tyr c-2J/ Tyr c-2J and the C3H
progenitor strains. F1 progeny were backcrossed once to the B6 strain prior to
intercrossing for 20 generations. All F20+ mice are considered homozygous
and contain ~75% of their alleles from B6 and ~25% of their alleles from
C3H. We donated BXH22 and B6cC3-1 to The Jackson Laboratory (http://
The TJU Animal Facility provides a specific pathogen-free environment
and is accredited by the Association for the Assessment and Accreditation
of Laboratory Animal Care (AAALAC). Mice were housed in polycarbonate
cages from Allentown Caging Equipment Co. (Allentown, NJ). Cages were
lined with alpha dry bedding from Shepherd Specialty Papers Inc (Kalamazoo,
MI). Mice were fed Laboratory Autoclavable Rodent Diet 5010 from Animal
Specialties and Provisions (ASAP) (Quakertown, PA). Food, cages, bedding,
and filtered water were autoclaved before use. All protocols were approved by
the TJU IACUC committee.
F1 offspring were produced by crossing BXH# RI females to B6 ApcMin/+
males. Offspring were genotyped for the ApcMin mutation (see DNA isolation
and genotyping). Of the offspring produced from this mating, Apc+/+ females
were mated to B6 ApcMin/+ males, creating the N2 generation (Supplementary
Figure 1, available at Carcinogenesis Online). Offspring that carried ApcMin
were subsequently analyzed for polyp number, size, and location along the
intestinal tract. This backcross protocol was repeated to produce successive
DNA isolation and genotyping
At weaning, tail biopsies of ~0.5 cm were placed in 1.5 ml Eppendorf tubes
and stored in ice for DNA isolation. Each mouse was ear-notched for
identification. DNeasy (Qiagen, Valencia, CA) isolation kits were used to extract
genomic DNA from tail tissue according to the manufacturer’s protocol. Mice
were genotyped for ApcMin by PCR analysis as described (
Polyp counting procedure
ApcMin/+ mice were aged for 110–130 days, then sacrificed via CO2
asphyxiation followed by cervical dislocation. The intestinal tract was removed and
cut longitudinally. The small intestine was divided into proximal (psi),
middle (msi), and distal (dsi) sections. The colon was divided into proximal
(pc) and distal (dc) sections. Each section was washed with 1× Dulbecco’s
phosphate-buffered saline (pH 7.0) to eliminate intestinal contents. A Nikon
SMZ-U dissecting microscope (×15 magnification) was used to visualize
polyps. For each section of the intestine, the number, size, and location were
recorded as described (
). Polyp size was measured using an optical
grid inserted into the microscope eyepiece.
Whole genome single-nucleotide polymorphism (SNP) genotyping
Genomic DNA from BXH14 ApcMin/+ N2 and N3 mice (along with B6,
C3H, and F1 control mice) were genotyped using the Illumina low-density
SNP panel (377 SNPs) at the Harvard Partners Center for Genetics and
Genomics (Cambridge, MA). Discriminating SNPs and four additional
markers (D1Mit206, D2Mit307, D2Mit285, and D18Kcc1) along with coat
color (agouti or non-agouti) were analyzed by the Chi-square rxc method
with Yates’ correction using GraphPad Software (http://www.graphpad.com/
Gene interaction network analysis
Gene interaction networks were identified using Ingenuity® Pathway Analysis
(IPA; Ingenuity® Systems, Redwood City, CA). Ensembl gene identifiers for
each gene in each region were obtained from the Ensembl database using
BioMart. Gene lists were uploaded into IPA and networks of highly
interconnected genes were derived from the Ingenuity Pathways Knowledgebase
following the provider’s protocol. The statistical significance of interaction
networks was determined by the Fisher’s exact test. P-values associated with
functional annotations and canonical pathways were corrected for multiple
testing using the false discovery rate controlling method of Benjamini and
Average polyp numbers in BXH RI ApcMin/+ mice vary from high
Although our previous studies had demonstrated that the C3H
genome contains modifier loci of the ApcMin mutation (
), we chose to
use an existing RI series to aid in the identification of these
resistant alleles. The progenitor inbred strains for the BXH RI lines are
C57BL/6J (B6) and C3H/HeJ (C3H). Females from the BXH# RI
lines were crossed to B6 ApcMin/+ males to produce F1 hybrid
progeny (Supplementary Figure S1, available at Carcinogenesis Online).
Five BXH RI lines (BXH2, BXH4, BXH8, BXH14, and BXH22)
and one BXH RC line (B6cC3-1) were tested for their
susceptibility or resistance to intestinal tumorigenesis. These lines were chosen
because they were homozygous for susceptible Mom1S alleles,
originally derived from the B6 strain, thus eliminating the influence of the
Mom1R locus from this study. The ApcMin/+ offspring from each group,
hereafter referred to as BXH# F1, were aged to 110–130 days and
scored for adenomas throughout the small intestine and colon.
The small intestinal polyp number for each parental control line
showed the expected phenotypic extremes (Table I). The high
control B6 ApcMin/+ group had an average of 66 ± 34 polyps, whereas
the low control C3H.B6 Mom1S/S ApcMin/+ group had an average of
10 ± 5 polyps. These data are consistent with results from our
previous studies (
Based on the average small intestinal polyp numbers (Table I), a
gradient in phenotype was observed from high polyp number to low
polyp number (B6cC3-1 > BXH2 > BXH4 > BXH22 > BXH8 >
BXH14). The average polyp number (60 ± 32) in the B6cC3-1 F1
ApcMin/+ offspring most closely resembled the average polyp number
(57 ± 27) in the high control B6 Mom1S/S ApcMin/+ group, whereas the
BXH14 F1 ApcMin/+ group exhibited an average polyp number (8 ± 3)
that was less than the average polyp number (10 ± 5) in the low control
C3H.B6 Mom1S/S ApcMin/+ group. Note that the BXH14 F1 ApcMin/+
group was unique in that it exhibited the lowest small intestinal polyp
numbers of all lines tested.
Based on the average colon polyp numbers (Table I), a gradient in
phenotype was observed from high polyp number to low polyp
number (B6cC3-1 > BXH4 > BXH22 > BXH8 > BXH2 > BXH14). The
average polyp number (0.70 ± 0.88) in the B6cC3-1 F1 ApcMin/+
offspring was second only to the average polyp number (1.65 ± 1.81) in
the high control B6 Mom1S/S ApcMin/+ group. The BXH14 F1 ApcMin/+
group exhibited the lowest average polyp number (0.11 ± 0.32), which
was less than the average polyp number (0.31 ± 0.59) in the low
control C3H.B6 Mom1S/S ApcMin/+ group. For colon polyp incidence
(Table I), a gradient in phenotype was observed from high colon
polyp incidence to low colon polyp incidence (B6cC3-1 > BXH4 >
BXH22 > BXH2 > BXH8 > BXH14). The colon polyp incidence for
each parental line exhibits the expected phenotypic extremes of the
high control B6 ApcMin/+ group (69%) and the low control C3H.B6
Mom1S/S ApcMin/+ group (25%). The B6cC3-1 RC line was second
only to the phenotype of the B6 ApcMin/+ strain. The BXH14 line was
the only Mom1S/S line that had a lower average colon polyp number
and incidence than the C3H.B6 Mom1S/S ApcMin/+ group (Table I).
Statistical analyses of polyp numbers reveal a unique BXH phenotype
The Student’s t-test was used to evaluate the average number of small
intestinal adenomas for each group of Mom1S/S ApcMin/+ F1 offspring
compared with the control groups (and each other) (Supplementary
Table SI, available at Carcinogenesis Online). ApcMin/+ F1 offspring of
the B6cC3-1 RC line do not significantly differ (P = 0.293) from the
high control B6 ApcMin/+ group, but do significantly differ (P < 0.001)
from the low control C3H.B6 ApcMin/+ group. Therefore, the B6cC3-1
RC line appears to have inherited susceptible B6 modifier alleles. In
contrast, F1 offspring from the BXH14 RI line have an average polyp
number that is significantly different (P < 0.001) from the high
control B6 ApcMin/+ group and is also significantly different (P = 0.045)
from the low control C3H.B6 ApcMin/+ group. In addition, the hybrid
BXH14 ApcMin/+ offspring significantly differ (P < 0.001) from all
other BXH RI and RC offspring tested (Supplementary Table SI,
available at Carcinogenesis Online). Therefore, the BXH14 RI line
appears to have inherited the greatest number of C3H modifier alleles
that protect against the development of polyposis in the presence of
the ApcMin mutation.
The Fisher’s exact probability test was used to evaluate colon polyp
incidence for each group of Mom1S/S ApcMin/+ F1 offspring compared
with the control groups (and each other) (Supplementary Table S2,
available at Carcinogenesis Online). ApcMin/+ F1 offspring from the
B6cC3-1 RC line do not significantly differ (P = 0.110) from the
high control B6 ApcMin/+ group. Therefore, this B6cC3-1 RC line
appears to have inherited susceptible B6 modifier loci. In contrast, F1
offspring from BXH2, BXH8, BXH14, and BXH22 lines significantly
differ (P < 0.005) from the high control B6 ApcMin/+ group, but not
the low control C3H.B6 ApcMin/+ group. However, the BXH14 RI line
is the only RI line that significantly differs from all but one of the
BXH RI lines (P < 0.05), but does not differ from the low control
C3H.B6 ApcMin/+ group. With a colon polyp incidence of only 11%,
the BXH14 RI line has the lowest colon polyp incidence of any
Mom1S/S ApcMin/+ F1 group. Similar observations were made with
respect to colon polyp number (Supplementary Table S3, available at
BXH14 ApcMin/+ F1 mice develop fewer polyps in each region of the
small intestine and colon compared with susceptible strains
The data demonstrate that the BXH14 RI genome protects against
intestinal polyposis in ApcMin/+ F1 hybrids. To determine whether
this decrease in polyposis was detectable along the entire small
intestine or was limited to a region of the small intestine, we
compared average polyp numbers from the resistant BXH14 ApcMin/+ F1
group to the susceptible B6cC3-1 ApcMin/+ F1 group; the B6cC3-1
group was chosen because as an RC line, it has the largest
number of B6 alleles of all BXH lines tested. The B6cC3-1 ApcMin/+ F1
group was most like the high control B6 ApcMin/+ group in phenotype
(Table I). BXH14 ApcMin/+ F1 mice develop an average of 0.5 ± 1.0,
2.9 ± 3.1, and 5.2 ± 3.6 polyps in the proximal, middle, and distal
portions of the small intestine, respectively (Figure 1A). The
resistant BXH14 ApcMin/+ F1 mice have significantly fewer (P < 0.0002)
polyps in all portions of the small intestine than the susceptible
B6cC3-1 ApcMin/+ F1 mice (6.1 ± 3.0, 19.5 ± 11.1, and 32.3 ± 18.4
for proximal, middle, and distal, respectively). Consistent with this
finding, the average polyp number for each portion of the small
intestine in the BXH14 ApcMin/+ F1 group is significantly different
(P < 0.0002) from the average polyp number of the high control B6
ApcMin/+ group (4.7 ± 3.0, 21.5 ± 13.3, and 40.2 ± 20.5 for proximal,
middle, and distal, respectively). Therefore, the BXH14 modifier
loci affect the entire length of the small intestine. Interestingly, the
average polyp number for the proximal (P = 0.0004) and middle
(P = 0.0116) small intestine is significantly lower in the BXH14
ApcMin/+ F1 group than the low C3H.B6 ApcMin/+ F1 control group
(0.4 ± 0.7, 2.7 ± 1.7, and 4.8 ± 2.5 for proximal, middle, and distal,
respectively), suggesting that the overall lower average observed in
the BXH14 strain compared with low C3H.B6 ApcMin/+ F1 control
group is primarily due to differences in the proximal and middle
Similar observations were made in the colon (Figure 1B). Significant
reductions in average polyp numbers were observed in both the
proximal (P = 0.011) and distal (P < 0.002) sections of the colon in the
BXH14 ApcMin/+ F1 group (0 and 0.1 ± 0.31 for the proximal and distal
colon, respectively) compared with the susceptible B6cC3-1 ApcMin/+
F1 group (0.1 ± 0.4 and 0.6 ± 0.8 for the proximal and distal colon,
respectively). Consistent with this finding, the average polyp
number for each portion of the colon for the BXH14 ApcMin/+ F1 group
is significantly different (P ≤ 0.0002) from the average polyp
number of the high control B6 ApcMin/+ group (0.6 ± 0.9 and 1.0 ± 1.6 for
the proximal and distal colon). Colon polyp incidence did not vary in
the proximal and distal sections between BXH14 ApcMin/+ F1 group
and the low control C3H.B6 ApcMin/+ F1 group (0 and 0.3 ± 0.6 for
the proximal and distal colon, respectively). Therefore, the BXH14
genome must harbor one or more modifier alleles of the ApcMin
mutation that act in a dominant fashion to protect against polyposis along
the length of the small intestine and colon.
Whole-genome SNP genotyping of N2 and N3 BXH14 ApcMin/+ mice
reveals Mom loci on Chrs 1, 2, 10, and 18
To identify C3H alleles that act to lower polyp number, the Illumina
low-density SNP panel was used to genotype 39 BXH14 ApcMin/+
mice from the N2 and N3 backcross generations across the genome
(Supplementary Figure S1, available at Carcinogenesis Online).
Mice were separated into a high group (n = 13) classified as
having >35 polyps, and a low group (n = 26) classified as having <35
polyps. A Chi-square rxc test was performed to test each SNP for
its prevalence within the high group versus the low group. The
results for SNPs that reached statistical significance are shown in
Supplementary Table S4, available at Carcinogenesis Online. Five
loci were found; in each case, the SNPs that reached the highest
statistical significance were C3H alleles. Because these SNPs were
flanked by C3H alleles, the low polyp phenotype most likely
originated from the C3H genome. These five loci are located on Chrs 1,
2 (two loci), 10, and 18 (Figures 2A–D, respectively); the loci were
given Modifier of Min # symbols for Mom14 on Chr 1, Mom15 on
Chr 2A (proximal), Mom16 on Chr 2B (distal), Mom17 on Chr 10,
and Mom18 on Chr 18.
The quantitative effect of the Mom14–18 loci on polyp number was
assessed using the marker with the lowest P-value at each Mom locus
(Supplementary Table S5, available at Carcinogenesis Online) and
calculating the average number of polyps for mice homozygous for the B6
allele versus mice heterozygous for B6 and C3H alleles (Supplementary
Table S5, available at Carcinogenesis Online). The data show that when
each Mom# locus is considered individually, average polyp number is
decreased by 39% for Mom14, 51% for Mom15, 46% for Mom16, 39%
for Mom17, and 52% for Mom18 in the heterozygous B6/C3H group
compared with the homozygous B6/B6 group. However, when we
compared these loci in pairwise combinations, polyp number in the small
intestine is decreased by 50–71% (Figure 2E; Supplementary Table SI,
available at Carcinogenesis Online). Furthermore, when combinations
of three Mom14–18 loci are analyzed together, average polyp number
was lowered by 58–75% in the heterozygous B6/C3H group compared
with the homozygous B6/B6 group. Regardless of the combination,
inheritance of four or more C3H alleles at the Mom14–18 loci resulted
in a 61–72% decrease in polyp number.
Based on the increasing level of protection afforded by the
combination of all five Mom14–18 loci (Figure 2E), we asked the question
of whether topologically defined groups of genes within the Mom14–
18 intervals had known interactions. To answer this question, we
turned to gene interaction network analysis using Ingenuity Pathway
Analysis (IPA) (www.ingenuity.com). Several possible outcomes
could result from this type of analysis of gene–gene interactions:
(i) no high scoring networks are generated indicating an absence
of gene–gene interactions, (ii) high scoring networks are found that
primarily contain genes located in a single Mom# region, or (iii)
high scoring networks are found that contain genes located in all
five Mom# regions. Remarkably, the highest scoring networks were
generated when genes located within all five Mom# regions were
evaluated together. The top five networks of genes identified by gene
interaction network analysis using IPA are shown in Supplementary
Table S6 and Figure S2, available at Carcinogenesis Online. These
significant P-values are interpreted as known gene–gene interactions
that are characteristic of each ensemble of genes. Every network
shown contains genes located within all five Mom# loci (Mom14,
Mom15, Mom16, Mom17, and Mom18), clearly indicating that there
are multiple possibilities by which gene–gene interactions could
BXH14 contains a unique combination of C3H alleles
The question of whether the C3H haplotype of the BXH14 line was
present in other BXH RI lines was answered using The Phenome
Database (www.jax.org/phenome). Haplotypes of the Mom14–18
loci were assessed for the BXH2, BXH4, BXH8, BXH14, BXH22,
and B6cC3-1 lines (Table II). The BXH14 RI line is the only line
tested that contains C3H alleles at all five resistant Mom14, 15, 16,
17, and 18 loci. Consequently, BXH14 exhibited the lowest average
small intestine polyp number and the lowest colon polyp incidence
of all BXH RI and RC lines tested. The BXH8 RI line contains C3H
alleles for Mom15 and Mom16, but has C3H alleles for only a
portion of Mom14, Mom17, and Mom18. Based on these findings, we
would predict that the BXH8 RI line would have low polyp numbers;
Table II shows that this prediction holds true in that the BXH8 F1
offspring had the second lowest average polyp number (28 ± 14) in
the small intestine as well as the second lowest colon polyp incidence
(24%) (second only to the BXH14 RI line). The remaining BXH2,
BXH4, BXH22, and B6cC3-1 lines contain only B6 alleles at two or
more Mom14–18 loci, and consequently exhibited higher polyp
numbers than BXH14 and BXH8.
Criteria to identify Mom14–18 genes
One strategy to find modifier genes is to examine genes implicated
in human colon cancers. We compared the Mom14–Mom18 loci to
studies identifying genes either (i) mutated in CRC, (ii) found through
genome-wide association studies (GWAS), and (iii) identified as
common insertions sites through transposon-mediated gene tagging
in the mouse (Table III). Each Mom14–18 locus contains genes that
(i) have been shown to influence human CRC susceptibility and/or
(ii) are mutated in human tumors. The genes that are mutated in CRC
were identified by DNA sequencing of human tumors, these genes
would presumably act in a cell-autonomous fashion. However, the
approach we described is unbiased, and therefore, will detect modifier
loci that can act in both non-cell or cell-autonomous fashion with
respect to the tumor lineage (Table III).
We identified 20 human genes that are located within the Mom14–
18 loci. Of these 20 genes, two of the mouse orthologs of these human
genes, Fmn1 and Lama5, contain non-synonymous coding (Cn) SNPs
that differ between B6 and C3H. The SNP differences between B6
and C3H may have functional impacts on gene products. However,
other types of variants in the Mom14–18 regions may also influence
phenotypes in ApcMin/+ mice. Additionally, common insertion sites
in 5 of these 20 genes (Eif3j, Dstn, Csnk2a1, Ube2n, and Zfp397)
have been observed in gastrointestinal tract tumors (
this approach has identified several candidate genes for further
characterization, other genes may be responsible for the effect of the
One expectation to account for the increased protection from
polyposis afforded when multiple modifier loci are present in a
single mouse (Figure 2E) is that modifier genes encoded by different
Mom# loci may interact with each other to alter phenotypes. Gene
interaction network analysis using IPA provides one means to
elucidate these networks. Supplementary Table S6 and Figure S2,
available at Carcinogenesis Online, show a relevant observation from this
study, namely that the five highest scoring functional networks
contain genes from all five Mom# loci (Mom14–18). Thus, we have clear
evidence from this approach that there are gene–gene interactions that
encompass all five Mom# loci.
To highlight candidate genes, we conducted a screen to identify genes
within the five IPA networks that contained either non-synonymous
coding SNPs or structural variants (insertion-deletions) between
the B6 and C3H strains (Supplementary Table S7, available at
Carcinogenesis Online). Remarkably, 18 of the 70 genes that interact
within the top network exhibit non-synonymous coding SNPs (www.
jax.org/phenome; Sanger1 and Sanger2 data sets). Networks 2–4 also
contain 13–14 genes that have non-synonymous coding changes.
Certainly, non-synonymous coding changes are only one type of
alteration that could impact gene function. Other genomic, as well as
epigenetic, changes could impact the spatial, temporal, and level of
gene expression leading to changes in phenotype.
This study of the BXH RI lines has revealed five new modifier loci
that influence intestinal tumorigenesis in mice carrying the ApcMin
mutation. The loci were named Mom14 on Chr 1, Mom15 on Chr
2A (proximal), Mom16 on Chr 2B (distal), Mom17 on Chr 10, and
Mom18 on Chr 18. Of the six BXH RI lines tested, the BXH14 RI line
had the most protective effect on phenotype, in that both the small
intestinal polyp number and the colon polyp incidence were lower
than the low control (C3H.B6 Mom1S/S X B6 ApcMin/+) F1 group. This
unique phenotype was recapitulated in subsequent N2 and N3
backcross generations of carrier females mated to B6 ApcMin/+ males.
The BXH14 RI strain contains a rare combination of C3H regions
Overall, BXH14 contains modifier loci with the strongest protective
effect against polyposis of all the RI and RC lines tested (Table I). The
finding of modifier loci was possible because of the rare combination
of C3H alleles that had become fixed in the BXH14 RI line. Because
the first step in the generation of an RI line is an intercross between two
progenitor strains (B6 and C3H), the F1 progeny inherit half of their
alleles from each progenitors’ genome. The second step is an intercross
of F1 × F1 to yield the F2 generation. The probability that a single F1
offspring will pass five unlinked loci to their offspring is 1/64 (1/2n,
where n = the number of loci). In addition, the probability of
selecting two F1 mice that each carry all five loci Mom14–18 from the C3H
genome and are mated together is 1/64 × 1/64, or 1/4096. Note that this
calculation does not take into account the probability of fixing all five
C3H loci in the BXH14 RI line, as the F# intercrosses continue to F20 (a
homozygous inbred RI line). However, the probability is slightly higher
because two of the modifier loci are linked (Mom15 and Mom16).
The identification of modifier loci was possible because of DNA
polymorphisms present in the genomes of the B6 and C3H inbred
aGenes listed can be found in the dataset from (
bGenes listed can be found in the NHGRI: A Catalog of Published Genome-Wide Association Studies for the disease/trait colorectal cancer
cGenes listed can be found in the datasets as listed (
dGenes listed can be found in The Phenome Database (www.jax.org/phenome).
eThe association of AURKA with human colon cancer as described (47).
fGenes listed directly flank rs961253 (www.ensembl.org).
gGenes listen are all in close proximity to rs4778594 (
strains. Because the initial cross was to B6 ApcMin/+ males, it was
possible to follow the inheritance of C3H alleles. Each of the Mom14–18
loci has its peak within a region of C3H origin. Thus, it is predicted
that the resistant modifier genes should resemble alleles in their C3H
progenitor strain. Several possibilities exist for the decreased
average polyp numbers detected in the BXH14 strain compared with the
progenitor C3H.B6 strain. First, the BXH RI line may have fixed
unique mutations/natural variants that arose over time as the line was
established. Second, the combinations of chromosomal regions from
C3H and B6 present in the BXH14 strain may have affected epistatic
relationships between loci, possibly unmasking protective alleles,
which now contribute to the resistant phenotype. Third, the BXH14
phenotype could be the result of a combination of C3H alleles and
new allelic variants. If such a variant arose within a large
contiguous region inherited exclusively from the B6 progenitor, it would not
have been detectable in our assay. Thus, genomic sequencing of the
BXH14 will help distinguish among these possibilities. Further
studies are underway to distinguish among these possibilities and identify
the Mom14–18 genes.
Advantages of screens using RI strain sets
Susceptibility loci for intestinal and CRC have been localized to more
than half of all mouse chromosomes (Figure 3). Chr 18 has the
highest number of loci (five), while Chrs 2 and 4 have four loci each.
Mom14, Mom15, Mom16, and Mom17 map to unique chromosomal
locations, whereas Mom18 overlaps with Mom3 (Figure 3). Of the 31
loci shown, the causative genes have been identified for only three loci
(Mom1 = Pla2g2a, Mom2 = Atp5a1, and Scc1 = Ptprj) (
Although the conceptual strategy for moving from a locus to a gene
appears straightforward, several difficulties can be encountered that
hinder the identification process. The usual strategy is to establish and
evaluate a large quantitative trait loci (QTL) cross (backcross or
intercross) between two strains that exhibit the extremes of a phenotype.
Once the genotype and phenotype of offspring have been analyzed,
congenic lines containing the potential QTL loci are established.
However, success is dependent on the quantitative impact of the loci
on the phenotype itself (
). If the original phenotype results from the
combined action of several unlinked loci, then the probability of
recapitulating that phenotype within offspring decreases as the number of
loci involved increases.
The use of RI lines may provide an example of how this impediment
can be overcome. The strategy would utilize an identity-by-descent
approach, where offspring are genotyped and phenotyped at every
backcross generation to ensure that the loci responsible for the
phenotype are still present in a subset of offspring. In the case of the BXH14
RI line, the initial test of phenotype was performed in a one-step
backcross, and the confirmation of phenotype was performed with only
two additional backcross generations, thus expediting the process of
finding loci compared with large-scale backcrosses and intercrosses.
High-throughput sequencing in combination with continued
backcrosses to refine the Mom regions can be used to distinguish which
genes within each of the Mom14–18 regions are the best candidates
for further study.
Network analysis highlights candidate modifier genes
The finding of five non-overlapping networks each composed of
different genes from all five Mom# regions opens the door to the
possibility that more than one network acts to influence tumor phenotypes.
Each IPA network score indicates the likelihood that the assembly
of a set of focus genes in a network could be explained by random
chance alone. A score of 92, such as that observed for the top scoring
Network 1, translates into a 1 × 10−92 probability that the focus genes
in Network 1 are together due to random chance (Supplementary
Table S7, available at Carcinogenesis Online). Therefore, the
combination of genes in Network 1 is highly significant, supporting the
utility of our approach. In addition, every gene in Network 1 is located
within the Mom14–18 regions.
Interestingly, the top scoring network generated from this unbiased
analysis centers on hepatocyte nuclear factor 4 α (Hnf4α), a gene that is
essential for normal development of the mouse colon (Supplementary
Table S6 and Figure S2A, available at Carcinogenesis Online) (
Loss of Hnf4α in the gut influences inflammatory homeostasis and
affects the balance of differentiation between epithelial and goblet
). Tissue-specific knockouts of Hnf4α in ApcMin mice
cause suppression of polyposis via a decrease in expression of genes
within the oxidoreductase system (42). Furthermore, none of these
oxidoreductase target genes were located within the Mom14–18 loci,
suggesting that regulation of reactive oxygen species is not the sole
mechanism responsible for suppression of polyposis. Further
studies are needed to decipher the specific pathways within the networks
identified by IPA (Supplementary Table S6 and Figure S2, available at
Carcinogenesis Online) that are associated with intestinal and CRC.
The level of protection against polyposis is comparable regardless
of the combination of three or more Mom# loci (Figure 2E). Our
findings suggest a novel paradigm whereby resistance is determined by
varying combinations of a set of protective alleles in different
individuals. Reanalyzing GWAS data sets using additive statistical
models, in which the aggregate effects of SNPs are taken into account,
may have strong implications for the study of human disease risk.
However, individual genes, with relatively small effects, such as
lowering polyp number by 40%, may not have a strong enough effect to
be detected by GWAS (
). Therefore, mouse models can inform
GWAS, leading to hypothesis-driven screens for loci interacting in
an additive fashion (45). Our results highlight the power of using the
mouse to inform studies of human cancer.
Supplementary Tables 1–7 and Figures 1 and 2 can be found at
Professor Fredric Rieders Ph.D. Scholarship from the Fredric Rieders
Renaissance Foundation to S.C.N.
The gene symbols, Mom14–18, were approved for use by Lois Maltais and the
International Committee on Standardized Genetic Nomenclature for Mice (www.
informatics.jax.org/mgihome/nomen/). We thank Terry Hyslop and Edward
Pequignot for aid with statistical analysis, Sankar Addya, Joseph Brunner, Judith
Morgan for technical assistance, and Richard Crist, Carlisle Landel, Jacquelyn
Roth, Xiang Wang, and Charlene Williams for critical review of the manuscript.
Conflict of Interest Statement: None declared.
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