PMC

Pedigree and neuroimaging findings of consanguineous Indian kindred. (a) Family pedigree. The parents are first cousins with two affected children (individuals II-1 and II-3) and one healthy daughter (individual II-2). (b and c) Brain MRI of individual II-1 at age 16 months. T2 axial images show increased extra-axial cerebrospinal fluid spaces, large sulci (white arrows), bright signal in putamen (red line arrow), and delayed myelination. (d and e) Brain MRI of individual II-1 at age 5 years, 11 months. As seen on the same sequences and cuts as shown at age 16 months, there is persistence of the signs of atrophy (white arrows), resolution of the T2 signal change in the putamena, and improved but still decreased myelination. (f) Brain MRS of individual II-1 at age 5 years, 11 months shows low N-acetylaspartate (NAA) peak at approximately 2 parts per million in a voxel placed over the basal ganglia

Functional validation of AGC1 mutation in study proband’s cells. (a) Transport assays of wild-type and mutant AGC1. Time courses of [¹⁴C]aspartate/aspartate (panel A) and [¹⁴C]glutamate/aspartate (panel B) exchanges in proteoliposomes reconstituted with the recombinant wild-type (filled circles) or R353Q mutant AGC1 from individual II-1 (filled squares) are shown. At time zero, 50 μM [¹⁴C]aspartate (A) or 200 μM [¹⁴C]glutamate (B) was added to proteoliposomes containing 20 mM aspartate. At the indicated times, the uptake of the labeled substrate was stopped by the addition of 15 mM pyridoxal 5′-phosphate and 10 mM bathophenanthroline. Data shown indicates mean and standard deviation of four independent experiments that were each performed in duplicate. (b) Expression analysis of AGC1 in fibroblasts from AGC1 proband and unrelated healthy control. Mitochondrial proteins (40 μg) were separated on 15 % SDS-PAGE, transferred onto nitrocellulose membrane, and immunodecorated with anti-AGC1 (upper panel) or anti-porin (lower panel) antibodies. Densitometry analysis revealed similar content of AGC1 and porin in the investigated mitochondrial extracts in three independent experiments

(a) Overview of candidate genes with potentially pathogenic alleles identified by whole exome sequencing in one affected sibling (individual II-3). NS, non-synonymous variant. SS, acceptor or donor splice site variants. I coding indels, MAF minor allele frequency. ESP6500SI, 6503 exomes data from the NHLBI Exome Sequencing Project. *, Neither homozygous nor compound heterozygous variants were present in over 1,200 exome datasets that we had previously sequenced. (b) Details of two candidate genes in which homozygous missense variants were identified by whole exome sequencing in individual II-3. (c) Mutation validation by Sanger sequencing confirmed the SLC25A12 mutation (c.1058G>A, black arrow) segregates with disease in individuals II-1 and II-3. Their healthy sibling and parents are each heterozygous carriers, consistent with autosomal recessive disease. (d) Evolutionary conservation of the Arg353 residue in the AGC1 protein. The Arg353 residue is located just below the m-gate of the AGC1 carrier, where it participates in closing and opening of the carrier on the mitochondrial matrix side through an interaction with a highly conserved glutamate at residue 384. Hs Homo sapiens, Bt Bos taurus, Mm Mus musculus, Gg Gallus gallus, Dr Danio rerio, Xl Xenopus laevis, Ce Caenorhabditis elegans, Dm Drosophila melanogaster, Ag Anopheles gambiae, Nv Nasonia vitripennis, Sc Saccharomyces cerevisiae, Af Aspergillus fumigatus, Nc Neurospora crassa