MALDI-MSI Approach to Renal Biopsies of Patients with Fabry Disease

INTRODUCTION AND AIMS: Fabry disease (FD) is a X-linked hereditary condition due to the mutation of the enzyme alpha-galactosidase A with consequent intracellular accumulation of its substrate (Gb3). Fabry nephropathy (FN, Fig.1) can progress to ESRD if not adequately recognized and treated. Different mutations can determine different phenotypes (classic and atypical). Moreover, differences exist among males and females in terms of severity and time of onset of the disease, probably due to the Lyon effect. MALDI-MSI is a proteomic technique that analyzes in situ FFPE renal biopsies. METHODS: Renal biopsies from 6 patients with FN (2 males and 4 females) with different mutations (4 classic and 2 atypical) were firstly analyzed on LM and EM to assess the severity score (Fogo 2009 NDT) (Tab.1,2). For every patient, a 4-μm thick section from the corresponding FFPE block was cut and mounted onto a ITO slide. MALDI-MSI analysis was performed in reflectron positive mode in the mass range of m/z 750 to 2500 with the UltrafleXtreme MALDI-TOF/TOF MS. Images were acquired with a laser diameter and raster of 50 µm, data elaboration was performed as previously described (Smith BBA 2016). RESULTS: The only statistically relevant difference was higher prevalence of no sclerotic glomeruli in the female group (Tab. 2). Proteomics showed the absence of differences of spectra between females (Figure 2a, red) and males (Figure 2a, blue, PCA). On the other hand, differences in terms of protein profiles were found comparing classic (Figure 2b, blue) and atypical variants (Fig.2b, red), allowing the identification of 6 different putative ions up-regulated in both groups (AUC≥ 0,8). These findings were further confirmed by PCA with partial segregation of the spectra derived from the two groups. CONCLUSIONS: The present study demonstrated the feasible application of MALDI-MSI in the analysis of FN renal biopsies. Moreover, starting from the different clinical behaviour of the disease among males and females, the histological and proteomic analysis demonstrated the absence of differences between the two groups. Finally, the comparison of classic and atypical variants showed differences in terms of protein expression that can be used to discriminate these patients in the clinical setting. However, due to the rarity of this condition, one of the limitation of the present study is represented by the few cases analysed. For this reason, the analysis of further specimens is needed to confirm these findings and introduce them in the routine practice