Clinical Evaluation of Three KRS Families and Cellular Analysis of Distinct ATP13A2 Mutations Reveal Different Levels of Iron Accumulation.

Kufor-Rakeb Syndrome (KRS) is a rare neurodegenerative disease caused by homozygous mutations in the ATP13A2 gene. The ATP13A2 protein, found in lysosomal and late-endosomal membranes, performs cellular functions such as iron-chelating agent transport and intracellular iron homeostasis. Mutations in ATP13A2 can lead to intracellular iron accumulation; however, whether KRS caused by an ATP13A2 mutation falls under Neurodegeneration with Brain Iron Accumulation disorders has long been debated. The most fundamental reason is that magnetic resonance imaging (MRI) cannot identify iron deposits in the basal ganglia in all KRS cases. We hypothesize that analyzing iron deposition at the cellular level could be more sensitive in detecting varying levels of iron accumulation associated with different ATP13A2 mutations, and it may be more useful when conventional MRI fails to detect iron, yields inconclusive results, or cannot be performed. We identified two new ATP13A2 mutations (p.Leu518_Thr519del, and p.Leu939Pro) in this study and comparatively investigated the impacts of three distinct ATP13A2 mutations (p.Pro474fs, p.Leu518_Thr519del, and p.Leu939Pro) using KRS patients' primary fibroblasts and MCF7 cells overexpressing these mutated ATP13A2 proteins to analyze if these different mutations of ATP13A2 can cause differing levels of iron accumulation. Following the detection of iron deposits via Prussian blue staining and inductively coupled plasma mass spectrometry, the cell viability was assessed via MTT assay to ascertain the impact of iron accumulation. Each type of ATP13A2 mutation led to iron accumulation; however, frameshift and deletion mutations resulted in more iron accumulation than the missense mutation. In addition, the transient overexpression of the wild-type ATP13A2 attenuated the cell death caused by iron accumulation. This study demonstrated that different types of ATP13A2 mutations are related to varying levels of iron accumulation and provided an explanation for the inconsistent perspectives on the association of KRS with iron accumulation.