A novel method for quantification of globotriaocylceramide (GL-3) inclusions in affected podocytes in females with Fabry disease shows progressive accumulation of GL-3 in podocytes with age and no cross-correction between affected and non-affected podocytes

Background: While females can suffer serious FD complications, most studies are limited to males to avoid confounding the results by mosaicism phenomenon. We developed a method for quantifying GL-3 content of Fabry-affected podocytes in females independent of mosaicism. Methods: We applied mathematical approaches to morphometric data of 91 glomeruli from 40 untreated males with FD. We modeled the relationship between GL-3 inclusion volume density in podocyte [Vv(Inc/PC)] profiles with visible inclusions and the probability of obtaining random profiles without inclusions () and derived an equation for estimating Vv(Inc/PC) in females independent of mosaicism. The model was applied to biopsies from 10 females and 8 age-matched males with untreated FD. Mean podocyte volume (VPC) and total GL-3 inclusions volume [V(Inc/PC)] were estimated in affected podocytes in females. Results: A 4th degree polynomial equation best described the relationship between Vv(Inc/PC) and (R2=0.94) and passed k-fold cross-validation. VPC and V(Inc/PC) (r=0.87, p=0.002) were both increased with age in affected podocytes in females. Foot process width correlated with VPC (r=0.89, p=0.02) and V(Inc/PC) (r=0.87, p=0.02) . Comparisons with male biopsies showed no significant sex difference in Vv(Inc/PC) (p=0.75) or in V(Inc/PC) (p=0.45). Conclusion: We developed and validated a novel method for measuring GL-3 in Fabry podocytes independent of mosaicism in females. GL-3 accumulation and injury in Fabry podocytes in females is progressive with age. Importantly, GL-3 content in Fabry-affected podocytes in females is similar to that in males, consistent with absence of cross-correction between affected and non-affected cells. This method will be of great value for measuring podocyte GL-3 as an outcome variable in studies of females with FD.