SLC25A22 is a novel gene for migrating partial seizures in infancy

Abstract

Objective: To identify a genetic cause for migrating partial seizures in infancy (MPSI).

Methods: We characterized a consanguineous pedigree with MPSI and obtained DNA from affected and unaffected family members. We analyzed single nucleotide polymorphism 500K data to identify regions with evidence of linkage. We performed whole exome sequencing and analyzed homozygous variants in regions of linkage to identify a candidate gene and performed functional studies of the candidate gene SLC25A22.

Results: In a consanguineous pedigree with 2 individuals with MPSI, we identified 2 regions of linkage, chromosome 4p16.1-p16.3 and chromosome 11p15.4-pter. Using whole exome sequencing, we identified 8 novel homozygous variants in genes in these regions. Only 1 variant, SLC25A22 c.G328C, results in a change of a highly conserved amino acid (p.G110R) and was not present in control samples. SLC25A22 encodes a glutamate transporter with strong expression in the developing brain. We show that the specific G110R mutation, located in a transmembrane domain of the protein, disrupts mitochondrial glutamate transport.

Interpretation: We have shown that MPSI can be inherited and have identified a novel homozygous mutation in SLC25A22 in the affected individuals. Our data strongly suggest that SLC25A22 is responsible for MPSI, a severe condition with few known etiologies. We have demonstrated that a combination of linkage analysis and whole exome sequencing can be used for disease gene discovery. Finally, as SLC25A22 had been implicated in the distinct syndrome of neonatal epilepsy with suppression bursts on electroencephalogram, we have expanded the phenotypic spectrum associated with SLC25A22.

Figure 1

The affected boy (EP-201) and girl (EP-202) are shown as a black square and circle. The mother and father of the affected children (EP-205 and EP-206) were first cousins from Saudi Arabia. The unaffected brothers (EP-203 and EP-204) are shown as well.

Figure 2

The SNP genotype, plot of the LOD score, and schematic display of the genes in the regions are shown for the two regions with maximal LOD score in the EP-200 family. Part (a) depicts the region at chromosome 4p16.1-p16.3, and part (b) depicts the region at chromosome 11p15.4-pter. SLC25A22 is circled in red. The SNPs displayed as red and blue represent homozygous genotypes, whereas those shown in green represent heterozygous genotypes. (The ideograms for chromosomes 4 and 11 are derived from the UCSC Genome Bioinformatics website, www.genome.ucsc.edu.)

Figure 2

The SNP genotype, plot of the LOD score, and schematic display of the genes in the regions are shown for the two regions with maximal LOD score in the EP-200 family. Part (a) depicts the region at chromosome 4p16.1-p16.3, and part (b) depicts the region at chromosome 11p15.4-pter. SLC25A22 is circled in red. The SNPs displayed as red and blue represent homozygous genotypes, whereas those shown in green represent heterozygous genotypes. (The ideograms for chromosomes 4 and 11 are derived from the UCSC Genome Bioinformatics website, www.genome.ucsc.edu.)

Figure 3

(a) The chromatograms from Sanger sequencing confirmation of the homozygous c.G328C mutation are shown for EP-201 and EP-202. The unaffected siblings (EP-203 and E-204) and parents (EP-205 and EP-206) are all heterozygous, as seen by the two differently colored peaks and the denotation of “N”. (b) Amino acid conservation of two cerebral mitochondrial glutamate transporters, SLC25A22 and SLC25A18 (also known as GC1 and GC2), in humans and of SLC25A22 in several other species in the region of a novel SLC25A22 mutation associated with MPSI. The amino acid corresponding to the location of the p.G110R mutation is in bold.

Figure 3

(a) The chromatograms from Sanger sequencing confirmation of the homozygous c.G328C mutation are shown for EP-201 and EP-202. The unaffected siblings (EP-203 and E-204) and parents (EP-205 and EP-206) are all heterozygous, as seen by the two differently colored peaks and the denotation of “N”. (b) Amino acid conservation of two cerebral mitochondrial glutamate transporters, SLC25A22 and SLC25A18 (also known as GC1 and GC2), in humans and of SLC25A22 in several other species in the region of a novel SLC25A22 mutation associated with MPSI. The amino acid corresponding to the location of the p.G110R mutation is in bold.

Figure 4

(a) Overexpression of GFP-tagged WT SLC25A22 in COS-7 cells resulted in a punctate pattern of GFP, consistent with mitochondrial localization (green=GFP, blue=DAPI, red=mitochondrial marker MitoTracker Red CMXRos). There is widespread signal throughout the cytoplasm, outlining the boundaries of the cell, seen in the SLC25A22 panel. The middle panel shows labeling of mitochondria, and some colocalization of SLC25A22 and mitochondria is evident as yellow in the third panel, labeled Merge. (b) Transport assays of wild-type and mutant SLC25A22. At time zero, 1 mM [14C]glutamate was added to liposomes reconstituted with WT SLC25A22 or the G110R mutant SLC25A22 and containing 10 mM glutamate. After 1 and 60 minutes’ incubation the uptake of the labeled substrate was terminated by addition of 30 mM pyridoxal 5′-phosphate and 10 mM bathophenanthroline. The data represent the means ± S.D. of four independent experiments performed in duplicate.

Figure 4

(a) Overexpression of GFP-tagged WT SLC25A22 in COS-7 cells resulted in a punctate pattern of GFP, consistent with mitochondrial localization (green=GFP, blue=DAPI, red=mitochondrial marker MitoTracker Red CMXRos). There is widespread signal throughout the cytoplasm, outlining the boundaries of the cell, seen in the SLC25A22 panel. The middle panel shows labeling of mitochondria, and some colocalization of SLC25A22 and mitochondria is evident as yellow in the third panel, labeled Merge. (b) Transport assays of wild-type and mutant SLC25A22. At time zero, 1 mM [14C]glutamate was added to liposomes reconstituted with WT SLC25A22 or the G110R mutant SLC25A22 and containing 10 mM glutamate. After 1 and 60 minutes’ incubation the uptake of the labeled substrate was terminated by addition of 30 mM pyridoxal 5′-phosphate and 10 mM bathophenanthroline. The data represent the means ± S.D. of four independent experiments performed in duplicate.

Figure 5

The chief clinical features of MPSI and NEESBs are shown. A schematic representation of the SLC25A22 glutamate transporter protein, with a single reported protein domain, is shown with the MPSI-associated p.G110R mutation and the two NEESBs-associated mutations, p.P206L and p.G236W^, indicated with arrows.