Cdcs1 a major colitis susceptibility locus in mice; subcongenic analysis reveals genetic complexity

Abstract

Background: The cytokine-deficiency-induced colitis susceptibility (Cdcs)1 locus is a major modifier of murine inflammatory bowel disease (IBD) and was originally identified in experimental crosses of interleukin-10-deficient (Il10(-/-)) mice. Congenic mice, in which this locus was reciprocally transferred between IBD-susceptible C3H/HeJBir-Il10(-/-) and resistant C57BL/6J-Il10(-/-) mice, revealed that this locus likely acts by inducing innate hypo- and adaptive hyperresponsiveness, associated with impaired NF-kappaB responses of macrophages. The aim of the present study was to dissect the complexity of Cdcs1 by further development and characterization of reciprocal Cdcs1 congenic strains and to identify potential candidate genes in the congenic interval.

Methods: In total, 15 reciprocal congenic strains were generated from Il10(-/-) mice of either C3H/HeJBir or C57BL/6J genetic backgrounds by 10 cycles of backcrossing. Colitis activity was monitored by histological grading. Candidate genes were identified by fine mapping of congenic intervals, sequencing, microarray analysis, and a high-throughput real-time reverse-transcription polymerase chain reaction (RT-PCR) approach using bone marrow-derived macrophages.

Results: Within the originally identified Cdcs1-interval, 3 independent regions were detected that likely contain susceptibility-determining genetic factors (Cdcs1.1, Cdcs1.2, and Cdcs1.3). Combining results of candidate gene approaches revealed Fcgr1, Cnn3, Larp7, and Alpk1 as highly attractive candidate genes with polymorphisms in coding or regulatory regions and expression differences between susceptible and resistant mouse strains.

Conclusions: Subcongenic analysis of the major susceptibility locus Cdcs1 on mouse chromosome 3 revealed a complex genetic structure. Candidate gene approaches revealed attractive genes within the identified regions.

FIGURE 1

Genotype and phenotype of Il10^−/− Chr 3 interval-specific congenic mouse strains. Different subcongenic B6.C3Bir-Cdcs1 (BC; 1A & 1C) or C3Bir.B6-Cdcs1 (CB; 1B & 1D) lines (denoted as R1-R8), the markers used for genotyping with positions in mega base pairs (Mbp), cecum/colon total scores, numbers (N) of mice used for analysis, and detailed p-values (Fig. 1A–B) are shown. The candidate gene intervals determined by the respective congenic set of mice are highlighted in Fig 1A and 1B. Resistant (R), susceptible (S), intermediate (I) was defined based on results of the Median test, determining whether a given congenic strain differed from either the B6-Il10^−/− or the C3Bir-Il10^−/− parental strain or both. Figures 1C and 1D show typhlitis scores (single values and medians) of male or female B6-Il10^−/−, C3Bir-Il10^−/−, and BC-R7 (Fig. 1C) or CB-R6 (Fig. 1D) mice and p-values determined by the Newman-Keuls Multiple Comparison Test; ns = not significant; na = not applicable.

FIGURE 1

Genotype and phenotype of Il10^−/− Chr 3 interval-specific congenic mouse strains. Different subcongenic B6.C3Bir-Cdcs1 (BC; 1A & 1C) or C3Bir.B6-Cdcs1 (CB; 1B & 1D) lines (denoted as R1-R8), the markers used for genotyping with positions in mega base pairs (Mbp), cecum/colon total scores, numbers (N) of mice used for analysis, and detailed p-values (Fig. 1A–B) are shown. The candidate gene intervals determined by the respective congenic set of mice are highlighted in Fig 1A and 1B. Resistant (R), susceptible (S), intermediate (I) was defined based on results of the Median test, determining whether a given congenic strain differed from either the B6-Il10^−/− or the C3Bir-Il10^−/− parental strain or both. Figures 1C and 1D show typhlitis scores (single values and medians) of male or female B6-Il10^−/−, C3Bir-Il10^−/−, and BC-R7 (Fig. 1C) or CB-R6 (Fig. 1D) mice and p-values determined by the Newman-Keuls Multiple Comparison Test; ns = not significant; na = not applicable.

FIGURE 1

Genotype and phenotype of Il10^−/− Chr 3 interval-specific congenic mouse strains. Different subcongenic B6.C3Bir-Cdcs1 (BC; 1A & 1C) or C3Bir.B6-Cdcs1 (CB; 1B & 1D) lines (denoted as R1-R8), the markers used for genotyping with positions in mega base pairs (Mbp), cecum/colon total scores, numbers (N) of mice used for analysis, and detailed p-values (Fig. 1A–B) are shown. The candidate gene intervals determined by the respective congenic set of mice are highlighted in Fig 1A and 1B. Resistant (R), susceptible (S), intermediate (I) was defined based on results of the Median test, determining whether a given congenic strain differed from either the B6-Il10^−/− or the C3Bir-Il10^−/− parental strain or both. Figures 1C and 1D show typhlitis scores (single values and medians) of male or female B6-Il10^−/−, C3Bir-Il10^−/−, and BC-R7 (Fig. 1C) or CB-R6 (Fig. 1D) mice and p-values determined by the Newman-Keuls Multiple Comparison Test; ns = not significant; na = not applicable.

FIGURE 1

Genotype and phenotype of Il10^−/− Chr 3 interval-specific congenic mouse strains. Different subcongenic B6.C3Bir-Cdcs1 (BC; 1A & 1C) or C3Bir.B6-Cdcs1 (CB; 1B & 1D) lines (denoted as R1-R8), the markers used for genotyping with positions in mega base pairs (Mbp), cecum/colon total scores, numbers (N) of mice used for analysis, and detailed p-values (Fig. 1A–B) are shown. The candidate gene intervals determined by the respective congenic set of mice are highlighted in Fig 1A and 1B. Resistant (R), susceptible (S), intermediate (I) was defined based on results of the Median test, determining whether a given congenic strain differed from either the B6-Il10^−/− or the C3Bir-Il10^−/− parental strain or both. Figures 1C and 1D show typhlitis scores (single values and medians) of male or female B6-Il10^−/−, C3Bir-Il10^−/−, and BC-R7 (Fig. 1C) or CB-R6 (Fig. 1D) mice and p-values determined by the Newman-Keuls Multiple Comparison Test; ns = not significant; na = not applicable.

FIGURE 2

Subcongenic strain pair illustrating the Cdcs1.1 interval. Comparison of the highly susceptible BC-R2 and partially susceptible BC-R7 mice, the latter showing susceptibility in the cecum (males), indicates genetic modifiers in a region from 88.1 to 108.8 Mbp on Chr 3. Mbp = mega base pairs; S = susceptible; R = resistant; ex. = exon.

FIGURE 3

Subcongenic strain pair illustrating the Cdcs1.2 interval. Colon phenotypes of CB-R6 and CB-R8 mice suggest alleles that contribute to disease susceptibility between 120.0 and 123.0 Mbp on Chr 3. Mbp = mega base pairs; S = susceptible; R = resistant; ex. = exon

FIGURE 4

Subcongenic strain pair illustrating the Cdcs1.3 interval. Phenotypes of BC-R2 and BC-R8 mice indicate genetic factors that contribute to disease susceptibility between 125.6 and 128.0 Mbp on Chr 3. This candidate gene interval results from the presumption that BC-R8 and BC-R2 share common modifier; however, if BC-R8 mice carry independent modifier genes, a maximum of 1.3 Mbp (including the gene EG668828) have to be added to the candidate gene interval, as BC-R4 mice are resistant and carry C3Bir alleles at 129.3 Mbp. Mbp = mega base pairs; S = susceptible; R = resistant; ex. = exon