Natural mutations leading to down-regulation of Bbs7 in the Berlin Fat Mouse

Background and Aims The Berlin Fat Mouse Inbred (BFMI) line carries natural mutations that can help understanding genetic mechanisms causing obesity. The jObes1 locus on chromosome 3 in BFMI explains ~40% of the variance of body weight. The expression of the candidate gene Bbs7 is reduced in brain, adipose tissue and liver. The goal was to identify potential regulatory DNA variants.

Material and Methods To investigate the genetic effect on expression, we compared the effect of 16 DNA sequence variants between BFMI and C57BL/6N (B6N) in the promoter region and a 1578 bp deletion in intron 8 of Bbs7. Various sequences of the Bbs7 promoter of BFMI and B6N were cloned into dual-luciferase reporter plasmids. Plasmids were transfected into HEK cells in which the luciferase expression served as readout.

To evaluate the role of the deletion in intron 8, CRISPR/Cas9 mice were generated and crossed with mice carrying BFMI or B6N alleles. Complementation tests were performed on the offspring.

Results All plasmids containing the SNP rs29947545 in the 5‘ UTR of the Bbs7 promoter significantly reduced the expression of Bbs7 in HEK cells (0.35≤Fold Change ≤ 0.51, p≤0.042).

Complementation tests showed that CRISPR/Cas9 modified B6N mice partially complement (13.1 - 15.1%) the jObes1 allele which was accompanied by a trend towards a reduced Bbs7 expression in these mice.

Conclusion Both, a 5’ UTR-SNP and a deletion in intron 8 of Bbs7, contribute to the phenotype of the BFMI mice most likely by reducing Bbs7 expression.

15. Versorgungsforschung | Gesundheitsökonomie | Qualitätssicherung

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Artikel online veröffentlicht:
26. Mai 2022