Nava C et al. (2015), Hypomorphic variants of cationic amino acid tra...

XB-ART-53381

Amino Acids 2015 Dec 01;4712:2647-58. doi: 10.1007/s00726-015-2057-3.

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Hypomorphic variants of cationic amino acid transporter 3 in males with autism spectrum disorders.

Nava C , Rupp J , Boissel JP , Mignot C , Rastetter A , Amiet C , Jacquette A , Dupuits C , Bouteiller D , Keren B , Ruberg M , Faudet A , Doummar D , Philippe A , Périsse D , Laurent C , Lebrun N , Guillemot V , Chelly J , Cohen D , Héron D , Brice A , Closs EI , Depienne C .

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Cationic amino acid transporters (CATs) mediate the entry of L-type cationic amino acids (arginine, ornithine and lysine) into the cells including neurons. CAT-3, encoded by the SLC7A3 gene on chromosome X, is one of the three CATs present in the human genome, with selective expression in brain. SLC7A3 is highly intolerant to variation in humans, as attested by the low frequency of deleterious variants in available databases, but the impact on variants in this gene in humans remains undefined. In this study, we identified a missense variant in SLC7A3, encoding the CAT-3 cationic amino acid transporter, on chromosome X by exome sequencing in two brothers with autism spectrum disorder (ASD). We then sequenced the SLC7A3 coding sequence in 148 male patients with ASD and identified three additional rare missense variants in unrelated patients. Functional analyses of the mutant transporters showed that two of the four identified variants cause severe or moderate loss of CAT-3 function due to altered protein stability or abnormal trafficking to the plasma membrane. The patient with the most deleterious SLC7A3 variant had high-functioning autism and epilepsy, and also carries a de novo 16p11.2 duplication possibly contributing to his phenotype. This study shows that rare hypomorphic variants of SLC7A3 exist in male individuals and suggest that SLC7A3 variants possibly contribute to the etiology of ASD in male subjects in association with other genetic factors.

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Species referenced: Xenopus laevis
Genes referenced: mtor slc7a3 tomm20

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	Fig. 1. Identification of missense variants in SLC7A3 in four patients with ASD. a Pedigree of the families and segregation of SLC7A3 variants; m and m/+ denotes male or female individuals carrying one variant in the hemizygous or heterozygous state, respectively. Arrow the index case. Black symbols individuals diagnosed with ASD; gray symbols patients with undetermined intellectual disability or learning difficulty phenotypes (see details in Supplementary data). b Sequence electropherograms showing the SLC7A3 variants in the hemizygous state in the affected individuals of Families 505 (01, 02), 885, 388, 962 (P), in the heterozygous state in the mother of Family 505 (05) and their absence in an unaffected brother in Family 505 (03) and controls (C). c Alignment of the regions flanking the SLC7A3/CAT-3 missense variant in orthologous proteins showing the conservation of the altered amino acids. d Schematic model of the CAT-3 protein showing the putative location of the amino acid residues altered by the variants
	Fig. 2. Subcellular localization and expression of CAT-3 mutants at the plasma membrane in mammalian cells. a Subcellular localization of wild-type and mutant (p.Tyr430Cys, p.Ala331Thr, p.Ser589Thr, p.Ser595Thr) CAT-3 proteins and colocalization with endoplasmic reticulum (ER, marked using anti-calreticulin) observed by confocal microscopy. Scale bar 20 µm. b Representative western blot of WT and mutant CAT-3 protein expression in whole lysates and plasma membranes. Flotillin and Tom20 stainings were used to control membrane protein enrichment and normalize protein load, respectively. c Quantification of WT and mutant CAT-3 proteins present in whole lysates and plasma membranes. The values, obtained from at least three different experiments, were compared with the Mann–Whitney test; *p < 0.05
	Fig. 3. Analysis of WT and mutant CAT-3 transport activity, expression and localization in Xenopus laevis oocytes. a Transport of 100 µM [3H]L-arginine for 15 min into oocytes. b Representative Western Blot of WT and mutant CAT-3-EGFP proteins in whole cell lysates and at the plasma membrane. c Quantification of three independent experiments as shown in b, left columns CAT-3 in total cell lysate, right columns in plasma membrane protein fraction
	Fig. 4. Schematic diagram showing the possible consequences of SLC7A3 dysfunction on the mTOR and NO pathways. Reduced availability of intracellular cationic amino acids, including arginine, could decrease NO synthesis and alter NO-mediated signaling (on the left) or negatively affect the mTOR signaling pathway in neurons (on the right)

References [+] :

Amiet, Epilepsy in autism is associated with intellectual disability and gender: evidence from a meta-analysis. 2008, Pubmed