(586) ΔF508-CFTR mutation in C57BL/6 and Collaborative Cross mice to unravel the contribution of genetic background in the CF disease phenotype

Background: Several CF mouse models have been generated in the genetically-redundant inbred strains, such as C57BL/6. Recently, we introduced the Cftr-ΔF508 mutation of C57BL/6J (Cftr^tm1Kth) mice in the highly genetically diverse Collaborative Cross 037 (CC) and CC006 murine lines, selected from previous screenings. Thus, we aimed to explore whether and how the host genetic background in addition to CFTR mutation influence phenotypic traits.

Methods: We directly compared C57BL/6J and CC lines with Cftr-ΔF508 mutation for prenatal lethality, survival with laxative administration and salivary secretion after drug stimulation. Genomic content of CC037 and CC006 ΔF508/wt lines was explored by whole genome sequence (WGS) analysis after the backcross.

Results: WGS showed that the CC037 and CC006 lines shared 26,7% and 15,2% of the genome with C57BL/6J. The rest of the CC037 genome had haplotype contributions from NOD/ShiLtJ (27,9%), A/J (17,4%), 129S1/SvlmJ (10,5%), CAST/EiJ (4,8%) and WSB/EiJ (14%). For CC006, the haplotype contributions were from 129S1/SvlmJ (24,4%), NOD/ShiLtJ (21%), NZO/H1LtJ (13,9%), A/J (10,5%), PWK/phJ (5,1%), CAST/EiJ (5,5%) and WSB/EiJ (4,2%).

The heterozygotes ΔF508/wt of CC037, CC006 and C57BL/6J were normal and fertile, although the C57BL/6 and the CC037 line produced more pups per litter (6.4 pups, C57BL/6; 5.3 pups, CC037) than the CC006 line (3.5 pups). C57BL/6 ΔF508/ΔF508 mice, from heterozygous ΔF508/wt mouse crosses, were born at the expected Mendelian ratio (25,3%). Differently, breeding the heterozygous CC037 ΔF508/wt mice produced significantly fewer ΔF508/ΔF508 mice (14%) than expected. More dramatically, breeding the heterozygous CC006 ΔF508/wt mice did not produce ΔF508/ΔF508 mice (0%), indicating prenatal lethality.

The overall survival curve at 12 weeks showed significantly higher lethality for CC037 ΔF508/ΔF508 (86%) compared to C57BL/6 ΔF508/ΔF508 mice (38,5%). Males had significantly greater lethality compared to females in CC037 ΔF508/ΔF508, while C57BL/6 ΔF508/ΔF508 showed no gender difference. Interestingly, the laxative administration increased survival in the C57BL/6 ΔF508/ΔF508 but not in the CC037 ΔF508/ΔF508 mice, indicating an additional cause of mortality on top of gut obstruction. The CC037 ΔF508/ΔF508 mice showed endocardiosis with valve remodeling that could lead to cardiovascular disturbances.

Significative reduction in body weight was observed in ΔF508/ΔF508 of both CC037 and C57BL/6 genetic background compared to the relative wt/wt counterparts. However, CC037 ΔF508/ΔF508 mice were significantly smaller than C57BL/6 ΔF508/ΔF508, indicating a more severe phenotype.

Drug stimulation of salivary secretion revealed that the cAMP-dependent fraction was significantly decreased in ΔF508/ΔF508 mice compared to wt/wt for both CC037 and C57BL/6J lines. Differently, the Ca2+ stimulated component was lower in CC037 compared to C57BL/6J line, indicating that the contribution of other Cl- channel may be influenced by host genetic background.

Conclusions: By comparing novel CC lines with reference C57BL/6J mice, we showed that host genetic background in addition to CFTR mutations influence several phenotypic traits of CF pathology. We underline the need to explore additional genetic backgrounds in CF-disease models to unravel novel pathological phenotypes and open hypothesis for the CF pathology in humans.

Acknowledgements: Supported by grants from the Italian CF Research Foundation (FFC#4/2017 and FFC#2/2019) and CFF (BRAGON21I0).

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