Mutations in SLC25A22: hyperprolinaemia, vacuolated fibroblasts and presentation with developmental delay.

Mutations in SLC25A22 are known to cause neonatal epileptic encephalopathy and migrating partial seizures in infancy. Using whole exome sequencing we identified four novel SLC25A22 mutations in six children from three families. Five patients presented clinical features similar to those in the literature including hypotonia, refractory neonatal-onset seizures and developmental delay. However, the sixth patients presented atypically with isolated developmental delay, developing late-onset (absence) seizures only at 7 years of age. Abnormal metabolite levels have not been documented in the nine patients described previously. One patient in our series was referred to the metabolic clinic because of persistent hyperprolinaemia and another three had raised plasma proline when tested. Analysis of the post-prandial plasma amino acid response in one patient showed abnormally high concentrations of several amino acids. This suggested that, in the fed state, when amino acids are the preferred fuel for the liver, trans-deamination of amino acids requires transportation of glutamate into liver mitochondria by SLC25A22 for deamination by glutamate dehydrogenase; SLC25A22 is an important mitochondrial glutamate transporter in liver as well as in brain. Electron microscopy of patient fibroblasts demonstrated widespread vacuolation containing neutral and phospho-lipids as demonstrated by Oil Red O and Sudan Black tinctorial staining; this might be explained by impaired activity of the proline/pyrroline-5-carboxylate (P5C) shuttle if SLC25A22 transports pyrroline-5-carboxylate/glutamate-γ-semialdehyde as well as glutamate.

An optimised method for the proteomic profiling of full thickness human skin.

BACKGROUND: The skin is the largest organ of the human body and is the first line barrier defence against trauma, microbial infiltration and radiation. Skin diseases can be a result of multi-systemic disease or an isolated condition. Due to its proteolysis resistant properties there are relatively few human skin proteomic datasets published compared with other human organs or body fluids. Skin is a challenging tissue to analyse using traditional proteomic techniques due to its high lipid content, insolubility and extensive cross-linking of proteins. This can complicate the isolation and digestion of proteins for analysis using mass spectrometry techniques. RESULTS: We have optimised a sample preparation procedure to improve solubilisation and mass spectral compatibility of full thickness skin samples. Using this technique, we were able to obtain data for the proteome profile of full thickness human skin using on-line two-dimensional liquid chromatography, followed by ultra-high definition label-free mass spectrometry analysis (UDMS(E)). We were able to identify in excess of 2000 proteins from a full thickness skin sample. CONCLUSIONS: The adoption of on-line fractionation and optimised acquisition protocols utilising ion mobility separation (IMS) technology has significantly increased the scope for protein identifications ten-fold.