(581) Effects of Cftr genotype on circadian system function

Background: Sleep dysfunction is common among people with cystic fibrosis (CF). Reports indicate that disordered sleep in CF is consistent with circadian rhythm (CR) dysfunction. Little data exist on whether frequency and/or severity of CF-related sleep dysfunction are dependent upon Cftr genotype, though preliminary data from our clinic indicate that sleep dysfunction is common among CF patients of many genotypes. Data from our laboratory have shown that CF mice (F508del/F508del) exhibit altered clock gene expression profiles, circadian locomotor phenotypes, and serum melatonin levels when compared to WT controls. These findings provide a link between CR changes in CF patients and CR changes in CF mice. We have also published data suggesting microtubule instability as a mechanism underlying these changes. Previous findings from our lab indicate G542X and R117H human and mouse nasal epithelial cells also have impaired microtubule function, so testing CR phenotypes in CF mice of those genotypes would provide additional evidence supporting or refuting those data.

While we have established the validity of the CF mouse as a model system for studying CR dysfunction in CF, we have only examined F508del homozygotes. To establish clinical translatability of our findings, we must evaluate CR phenotypes in CF mice of diverse Cftr genotypes.

Methods: We evaluated CR-related phenotypes in CF mice of three genotypes: F508del/F508del, G542X/G542X, R117H/R117H. Each group was compared to WT controls in three categories: circadian locomotor activity, clock gene expression, and serum melatonin concentration. Circadian locomotor activity was measured for four weeks in 12/12 LD photoperiod and four weeks in DD photoperiod using surgically implantable telemetry devices. Clock gene expression and serum melatonin concentration were measured at four time points in 12/12 LD photoperiod. mRNA from suprachiasmatic nucleus (SCN) was examined via qPCR for the following clock genes: Clock, Bmal1, Period1, Period2, Cryptochrome1, and Cryptochrome2. Serum was extracted whole blood collected at sacrifice from each mouse and melatonin was measured using a standard melatonin ELISA kit. Melatonin and gene expression measurements were completed every six hours during the 12/12 LD photoperiod to establish a 24-hour pattern.

Results: Our investigation revealed significant differences in circadian locomotor activity, gene expression, and melatonin profiles in all three CF genotype groups compared to WT controls. These differences in F508del homozygotes are consistent with our previous findings in that genotype and reveal similar changes in mice homozygous for the G542X and R117H Cftr mutations.

Conclusions: These data replicate our publications showing a CR phenotype in CF mice homozygous for F508del and extend those observations to G542X and R117H homozygotes. These findings broaden the applicability of our CR studies to encompass diverse Cftr genotypes, supporting the claim that circadian system dysfunction is inherent to the loss or reduction of CFTR rather than functional consequence of a single mutation. Determining the impact of severe and mild Cftr mutations on CR phenotypes will provide information as to how much CFTR function needs to be restored to therapeutically to address issues relevant to patient quality of life.

Acknowledgements: This work was supported by the Cystic Fibrosis Foundation (BARBAT20F0, 2020).

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