Studying the Effect of the Host Genetic Background of Juvenile Polyposis Development Using Collaborative Cross and Smad4 Knock-Out Mouse Models

Abstract

Juvenile polyposis syndrome (JPS) is a rare autosomal dominant disorder characterized by multiple juvenile polyps in the gastrointestinal tract, often associated with mutations in genes such as Smad4 and BMPR1A. This study explores the impact of Smad4 knock-out on the development of intestinal polyps using collaborative cross (CC) mice, a genetically diverse model. Our results reveal a significant increase in intestinal polyps in Smad4 knock-out mice across the entire population, emphasizing the broad influence of Smad4 on polyposis. Sex-specific analyses demonstrate higher polyp counts in knock-out males and females compared to their WT counterparts, with distinct correlation patterns. Line-specific effects highlight the nuanced response to Smad4 knock-out, underscoring the importance of genetic variability. Multimorbidity heat maps offer insights into complex relationships between polyp counts, locations, and sizes. Heritability analysis reveals a significant genetic basis for polyp counts and sizes, while machine learning models, including k-nearest neighbors and linear regression, identify key predictors, enhancing our understanding of juvenile polyposis genetics. Overall, this study provides new information on understanding the intricate genetic interplay in the context of Smad4 knock-out, offering valuable insights that could inform the identification of potential therapeutic targets for juvenile polyposis and related diseases.

Keywords: Smad4; collaborative cross mice; intestinal cancer; intestinal polyps; juvenile polyposis syndrome.

Figure 1

Polyp counts in Smad4 heterozygous knock-out (KO) and wild-type (WT) mice. (A) The mean polyp count in the entire intestine is significantly higher in KO mice compared to WT mice (p < 0.001). (B) Variation in mean polyp count in the small intestine is significantly elevated in KO mice compared to WT mice (p < 0.001). (C) The mean polyp count in the colon is markedly higher in KO mice than in WT mice (p < 0.001) within the general mouse population. The X-axis represents the genotype, while the Y-axis represents the number of polyps. The statistical significance of differences in the average number of polyps between the two groups is presented as follows: (**) indicates a highly significant difference at p < 0.01.

Figure 2

Differential impact of Smad4 heterozygous knock-out on polyp counts in male and female mice. (A) Male mice with Smad4 heterozygous knock-out exhibit a significant increase in polyp count in the small intestine compared to wild-type mice (p < 0.001). (B) Female mice with Smad4 heterozygous knock-out demonstrate a significant increase in polyp count in the small intestine compared to wild-type mice (p < 0.001). (C) Smad4 heterozygous knock-out in male mice leads to a significant increase in polyp count in the colon (p < 0.001). (D) Female mice with Smad4 heterozygous knock-out show a significant increase in polyp count in the colon compared to wild-type mice (p < 0.001). (E) Smad4 heterozygous knock-out in male mice leads to a significant increase in polyp count in the whole intestinal tract (p < 0.001). (F) Female mice with Smad4 heterozygous knock-out also show a significant increase in polyp count in the whole intestinal tract compared to wild-type mice (p < 0.001). The X-axis represents the genotype, while the Y-axis represents the number of polyps. Statistical significance of differences in the average number of polyps between the two groups is denoted as follows: (**) indicates a highly significant difference at p < 0.01.

Figure 3

Comparison of polyp counts in F1 CC-C57BL/6 and F1 CC-C57BL/6 J-Smad4^tm1Mak lines. The average number of polyps (±SE) is shown for 14 F1 CC-C57BL/6 wild-type mice (blue-colored bars) and 14 F1 CC-C57BL/6 J-Smad4^tm1Mak heterozygous knock-out lines (orange-colored bars). The X-axis represents different collaborative cross (CC) lines, while the Y-axis shows the number of polyps in (A) the small intestine, (B) the colon, and (C) the entire intestinal tract. Statistical significance of differences in the average number of polyps between the two groups is denoted as follows: (*) indicates a significant difference at p < 0.05, and (**) indicates a highly significant difference at p < 0.01.

Figure 4

Correlation analysis of polyp development patterns in Smad4 heterozygous knock-out and wild-type mice populations. This figure presents the correlation analysis results for polyp development within the gastrointestinal tract of wild-type (WT) controls (A) compared to Smad4 heterozygous knock-out (KO) mice (B). The analysis reveals a significant positive correlation between the occurrence of Type C polyps in the colon and the presence of polyps in the small intestine within the KO population, suggesting a potential systemic effect or shared susceptibility factors. Furthermore, a marked positive correlation was observed between the total number of Type B polyps in the small intestine and the aggregate count of intestinal polyps in KO mice. This indicates that Type B polyps may be a predominant factor in the overall polyp burden. Additionally, a strong positive association is highlighted between the occurrence of Type B polyps in the small intestine and the total intestinal polyp count in KO mice, underscoring the significance of this polyp subtype in the observed pathology. These findings underscore the complex interplay between different polyp types in the intestines of Smad4 heterozygous knock-out mice and contribute to our broader understanding of polyp development dynamics in genetic models of intestinal tumorigenesis. The data include correlation coefficients and p-values, delineating statistical significance and facilitating a nuanced interpretation of polyp distribution and frequency patterns in relation to genetic modifications.

Figure 5

Gender-specific correlation patterns of polyp development in Smad4 heterozygous knock-out mice compared to wild-type mice. Correlation matrices for (A) KO female mouse lines, (B) WT female mouse lines, (C) KO male mouse lines, and (D) WT male mouse lines. In male KO mice, a positive correlation is evident between colon polyps and the C portion of small intestinal polyps. Additionally, a robust positive association exists between counts of total small intestinal B polyps and the overall number of intestinal polyps. In female KO mice, a positive correlation was observed between small intestinal polyps and small intestinal B polyps. Moreover, a positive correlation exists between total colon polyps and small intestinal C polyps. Notably, the previously observed positive correlation between total intestinal polyps and small intestinal A polyps in the KO group was absent in females. These gender-specific correlations provide valuable insights into the effects of Smad4 knock-out on polyp development, highlighting variations in the relationships between different polyp sizes in male and female mice.

Figure 6

Representative whole mounts of mouse intestine highlighting polyp classification. (A) Microscope’s magnification of the entire mounts stained to visualize polyps in a mouse with heterozygous Smad4 knock-out genotype. Depending on size criteria, multiple polyps are visible and categorized into three classes (A, B, and C). (B) Whole mount from a mouse with the wild-type genotype, showing fewer polyps in the same intestinal segment compared to the Smad4 knock-out mouse. The microscope’s magnification was 10×.