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

Abstract

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.

Fig. 1

Structural and ultrastructural examinations of SLC25A22-deficient fibroblasts. Fibroblast cell culture from control (a, e, i, m, q), patient 1 (b, f, j, n, r), patient 4 (c, g, k, o, s) and patient 5 (d, h, l, p, t). Ultrastructural examination by electron microscopy revealed widespread, almost exclusively empty vacuoles in all patients (a-h). Patients showed excess accumulation of lipid (i-l, Oil Red O; m-p, Sudan Black). Immunofluorescence of the autophagy marker p62 revealed increases in number and area of p62 punctae in patient cells, indicating a possible increase in autophagy or mitophagy due to mitochondrial dysfunction. Four representative images of p62 immunofluorescence per case were analysed using CellProfiler 2.1.1. All patient cells showed an increase in the mean number of p62 punctae per cell, which was significant in patient 4 (p = 0.002) (q). The mean area of p62 punctae in patient 1 was also increased compared to controls (r). Taken together, the mean total area of p62 staining per cell is increased in all patients compared to controls and significantly in patient 4 (p = 0.002) (s). Scale bars: a–e: 2 μm; f–h: 500 nm; i–p: 100 μm; q–t: 50 μm

Fig. 2

Brain magnetic resonance imaging of patients with SLC25A22 deficiency. (a, d and g) T2-weighted images. (b, c, f and h) T1-weighted images. (e and h) T1-weighted images with inversion recovery sequence. Patient 1 (2 y 1 m), patient 4 (6y 8 m) and patient 5 (2y 10 m). All patients show frontotemporal hypoplasia/atrophy (a, d and g) and prominence of cerebellar folia, consistent with cerebellar hypoplasia/atrophy (b, e and h). All patients also have a small splenium, with the splenium of the corpus callosum smaller than the genu and a small optic chiasma (smaller than the mammillary body) (c, f and i). Abnormal appearance of the insular cortex is also noted in patient 4 (d)

Fig. 3

Metabolism of proline and proposed additional transporter function of SLC25A22. Pathway showing the synthesis, degradation and interconversions of proline, P5C and related compounds within the mitochondria. 1, proline dehydrogenase; 2, P5C reductase; 3, ornithine δ-aminotransferase; 4, P5C synthase; 5, P5C dehydrogenase; 6, non-enzymatic; KA, keto-acid; AA, amino acid