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Identification of molecular mechanisms acting at the jObes1 locus in the Berlin Fat Mouse with effect on juvenile fat deposition

Subject Area Animal Breeding, Animal Nutrition, Animal Husbandry
Term from 2012 to 2023
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 226367462
 
Final Report Year 2024

Final Report Abstract

The Berlin Fat Mouse (BFMI) is characterized by the spontaneous occurrence of juvenile obesity. Crossbreeding experiments localized the highly significant recessive juvenile Obesity Locus 1. Fine mapping, complementation tests, and gene expression studies revealed that the Bardet-Biedl Syndrome 7 gene (Bbs7) is responsible for the obese phenotype. The BBS7 protein is essential for ciliary function, contributing to intracellular membrane receptor transport. Mutations in Bbs7 can cause Bardet-Biedl Syndrome in humans. The BFMI mouse also exhibits Bardet-Biedl Syndrome features, such as retinal impairment and brain structural changes in the brain, making this project relevant to humans. The aim was to elucidate the mechanism leading to Bbs7 dysfunction and juvenile obesity in BFMI. Data suggested that in BFMI mice, regulatory sequence variants rather than the BBS7 protein isoform could cause the defect in Bbs7 gene function. Genomic DNA deep sequencing did not uncover a significant mutation in the Bbs7 region in BFMI. Thus, the effect of individual mutations and haplotypes was examined. CRISPR/Cas9- modified mice lacking a 1,578 bp sequence with a CTCF element in Bbs7 intron 8, similar to BFMI, showed a significant but small impact on fat deposition. The deletion with the CTCF element only partially contributes to the obese phenotype and cannot solely explain the entire BFMI effect. Reporter gene assays identified rs29947545 as the only SNP reducing reporter gene expression by 60% when the BFMI allele is present. Since both the deletion and the BFMI SNP allele are found in other mice, it is suspected that another factor is likely required for normal Bbs7 expression, preventing obesity. However, complementation tests with BFMI showed no effect on Bbs7 haplotypes of mouse lines NZO, AKR, DBA/2J, and SJL, resembling widely the BFMI haplotype. HI-C analysis of three-dimensional DNA interactions in the Bbs7 region of BFMI and control animals showed no significant differences in astrocytes; however, expression analyses revealed ciliogenesis as significantly impaired biological process in BFMI astrocytes. These results suggest that at least two regulatory mutations are involved in reduced Bbs7 expression. BFMI may lack another factor preventing or compensating for faulty Bbs7 downregulation, thereby causing obesity. These findings contribute to a better understanding of non-coding sequence variants as causes of phenotypic variation.

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