Tools developed to help in the diagnosis of spastic paraplegia 56
CYP2U1 activity is altered by missense mutations in hereditary spastic paraplegia 56.
Hereditary Spastic Paraplegia (HSP) is an inherited disorder of the central nervous system mainly characterized by gradual spasticity and weakness of the lower limbs. SPG56 is a rare autosomal recessive early onset complicated form of HSP caused by mutations in CYP2U1. The CYP2U1 enzyme was shown to catalyze the hydroxylation of arachidonic acid. Here, we report two further SPG56 families carrying 3 novel CYP2U1 missense variants and the development of an in vitrobiochemical assay to determine the pathogenicity of missense variants of uncertain clinical significance. We compared spectroscopic, enzymatic and structural (from a 3D model) characteristics of the over expressed wild type or mutated CYP2U1 in HEK293T cells. Our findings demonstrated that most of the tested missense variants in CYP2U1 were functionally inactive because of a loss of proper heme binding or destabilization of the protein structure. We also showed that functional data do not necessarily correlate with in silico predictions of variants pathogenicity, using different bioinformatic phenotype prediction tools. Our results therefore highlight the importance to use biological tools, such as the enzymatic test set up in this study, to evaluate the effects of newly identified variants in clinical settings.
Figure: 3D-Model of the CYP2U1-AA complex, and location of the mutated residues in the
missense variants studied in this paper. Right view is deduced from left view by a 90° rotation around an axis perpendicular to the heme. Heme is in orange and AA in violet. Helixes F, G and I are shown in black; green and yellow, respectively. The BC-loop is shown in blue. The carbon atoms of mutated residues are indicated by red balls, the oxygen atoms by blue balls and the sulfur atoms by yellow balls.