Diet and Gender Influence Survival in Sickle Cell Disease

Increased childhood mortality and reduced longevity persist in the resource-limited settings with the highest populations of sickle cell disease (SCD) (Piel et al., New Engl J Med 2017). A high catabolic rate caused by multiple phenomena including compensatory erythropoiesis suggest increased calorie and nutritional requirements for individuals with SCD; however, a vast majority of individuals with SCD live under malnourished conditions. Also, it is unknown if the survival of offspring is influenced by the parents' diet and if the diet of individuals has a gender-specific effect on their survival.

To address this critical unmet need for information under controlled conditions, we performed a randomized double-blind trial on transgenic Berkley mice with SCD comparing an enriched 'sickle diet' (SD) with a standard 'rodent diet' (RD). The study was performed following protocols approved by the Institutional Animal Care and Use Committee. Breeder pairs comprised of hemizygous (HbAS) females crossbred with homozygous (HbSS) male mice were divided into two dietary groups: [i] SD (59M3, TestDiet; St. Louis, MO) and [ii] RD (9F, 5020, Harlan Laboratories; Madison, WI). SD contained 26.4% protein, 11.1% fat, 27.5% kcal from protein, and 26% kcal from fat. RD contained 18.6% protein, 6.2% fat, 24% kcal from protein, and 18% kcal from fat. SD had a higher mineral, vitamin, amino acid, and omega-3 fatty acid content than RD. Pups were weaned 3 weeks after birth and were then genotyped and phenotyped to select HbSS pups. Pups were then fed SD for 10 days and randomly assigned to either [i] SD or [ii] RD for the remainder of the study. Their survival was followed until the pups were 150 days of age. Analysis was performed on the relationship between rates of survival and the following factors of interest: pup gender, pup diet, and parental diet. The outcome variable of interest, survival in days, was examined using (1) Kaplan-Meier plots of estimated survival and (2) Cox regression to obtain hazard ratios (HR). For Cox regression, the significance of individual factors adjusted for multivariate data and the significance of overall differences were based on Wald tests and log-rank tests respectively. Data were analyzed using the package 'survival' in ' R 3.4.0 ' (R Foundation for Statistical Computing; Vienna, Austria). P <.05 was considered significant.

Of the 601 pups, 482 were on SD and 119 were on RD. After 150 days, the overall survival percentages of pups on SD and RD were 77% and 51% respectively. Pups fed SD had significantly prolonged survival rates compared to pups on RD when adjusted for multivariate data (p = 6.03*10^-8, HR: .3988, 95% confidence interval (CI): .2860 - .5561). Cumulatively, there were 338 female pups and 263 male pups. After 150 days, the overall survival percentages of female and male pups were 76% and 68% respectively. Gender significantly independently impacted survival with females showing greater survival than males when adjusted for multivariate data (p = .0125, HR: 1.4797, 95% CI: 1.0879 - 2.0126). After 150 days, the overall survival percentages of the 488 pups with parents on SD and the 113 pups with parents on RD were 73% and 68% respectively. Pups born to parents fed SD showed a trend towards increased survival; however, parental diet did not have a statistically significant impact on the survival of pups adjusted for multivariate data (p = .5392, HR: .8878, 95% CI: .6071 - 1.2982). Notably, male pups on RD born to parents on RD showed the poorest survival compared to all other groups, and male pups on SD born to parents on RD showed a significantly improved survival in comparison (p=0.0228), suggesting that improving the individual's diet can overcome the effect of the parents' diet.

A diet rich in protein, fat, and micronutrients significantly prolonged the survival of HbSS mice overall. HbSS females survived significantly longer than HbSS males. HbSS mice born to parents fed SD showed a trend towards increased survival; however, parental diet did not have a statistically significant impact on the survival of female pups. Overall, the data emphasized the importance of parents' diet, individual's diet, and gender in SCD. We speculate that an improvement of diet may reduce manifestations of SCD including pain, infections, inflammation, and organ damage and result in increased survival. These data also suggest the consideration of gender and diet in studies using mice with SCD to improve translational outcomes.