Little is known about the genetics of nonsyndromic intellectual disability (NSID). We hypothesized that de novo mutations (DNMs) in synaptic genes explain an important fraction of sporadic NSID cases. In order to investigate this possibility, we sequenced 197 genes encoding glutamate receptors and a large subset of their known interacting proteins in 95 sporadic cases of NSID. We found 11 DNMs, including ten potentially deleterious mutations (three nonsense, two splicing, one frameshift, four missense) and one neutral mutation (silent) in eight different genes. Calculation of point-substitution DNM rates per functional and neutral site showed significant excess of functional DNMs compared to neutral ones. De novo truncating and/or splicing mutations in SYNGAP1, STXBP1, and SHANK3 were found in six patients and are likely to be pathogenic. De novo missense mutations were found in KIF1A, GRIN1, CACNG2, and EPB41L1. Functional studies showed that all these missense mutations affect protein function in cell culture systems, suggesting that they may be pathogenic. Sequencing these four genes in 50 additional sporadic cases of NSID identified a second DNM in GRIN1 (c.1679_1681dup/p.Ser560dup). This mutation also affects protein function, consistent with structural predictions. None of these mutations or any other DNMs were identified in these genes in 285 healthy controls. This study highlights the importance of the glutamate receptor complexes in NSID and further supports the role of DNMs in this disorder.

Background and Purpose-We found previously that plasma levels of acrolein (CH 2 ϭCHCHO) and spermine oxidase (SMO) were well correlated with the degree of severity of chronic renal failure. The aim of this study was to test whether the levels of these 2 markers and of acetylpolyamine oxidase (AcPAO) were increased in the plasma of stroke patients. Methods-The activity of AcPAO and SMO and the level of protein-conjugated acrolein in plasma of the stroke patients and normal subjects were measured by high-performance liquid chromatography and ELISA, respectively. Focal infarcts were estimated by MRI or computed tomography (CT). Results-The levels of AcPAO, SMO, and acrolein were significantly increased in the plasma of stroke patients. The size of stroke was nearly parallel with the multiplied value of acrolein and total polyamine oxidase (AcPAO plus SMO). After the onset of stroke, an increase in AcPAO first occurred, followed by increased levels of SMO and finally acrolein. In 1 case, an increase in AcPAO and SMO preceded focal damage as detected by MRI or CT. Furthermore, stroke was confirmed by MRI in a number of mildly symptomatic patients (11 cases) who had increased levels of total polyamine oxidase and acrolein. Among apparently normal subjects (8 cases) who had high values of acroleinϫtotal polyamine oxidase, stroke was found in 4 cases by MRI. Conclusions-The

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In Figure 2B, the label on the right should be NR1-S560dup/NR2B instead of the alternative terminology NR1 (S560_T561InsS)/NR2B.

Properties of a membrane protein encoded byPolyamines, which are essential for cell growth, are regulated by biosynthesis, degradation, and transport (1-3). As for polyamine transport, properties of three polyamine transport systems were clarified by cloning the genes for these systems from Escherichia coli in our laboratory (4 -7). Two of them were spermidine-preferential and putrescine-specific uptake systems, in which ATP was necessary as the energy source. The third system was involved in the excretion of putrescine by a putrescine-ornithine antiporter activity. In eukaryotic cells,

The structure and function of the polyamine transport protein PotE was studied. Uptake of putrescine by PotE was dependent on the membrane potential. In contrast, the putrescine-ornithine antiporter activity of PotE studied with inside-out membrane vesicles was not dependent on the membrane potential (Kashiwagi, K., Miyamoto, S., Suzuki, F., Kobayashi, H., and Igarashi, K. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4529 -4533). The K m values for putrescine uptake and for putrescine-ornithine antiporter activity were 1.8 and 73 M, respectively. Uptake of putrescine was inhibited by high concentrations of ornithine. This effect of ornithine appears to be due to putrescine-ornithine antiporter activity because it occurs only after accumulation of putrescine within cells and because ornithine causes excretion of putrescine. Thus, PotE can function not only as a putrescine-ornithine antiporter to excrete putrescine but also as a putrescine uptake protein.Both the NH 2 and COOH termini of PotE were located in the cytoplasm, as determined by the activation of alkaline phosphatase and ␤-galactosidase by various PotE-fusion proteins. The activities of putrescine uptake and excretion were studied using mutated PotE proteins. It was found that glutamic acid 207 was essential for both the uptake and excretion of putrescine by the PotE protein and that glutamic acids 77 and 433 were also involved in both activities. These three glutamic acids are located on the cytoplasmic side of PotE, and the function of these three residues could not be replaced by other amino acids. Putrescine transport activities did not change significantly with mutations at the other 13 glutamic acid or aspartic acid residues in PotE.

We recently identified a gene (TPO1, YLL028w) that encodes a polyamine transport protein on the vacuolar membrane in yeast [Tomitori, Kashiwagi, Sakata, Kakinuma and Igarashi (1999) J. Biol. Chem. 274, 3265-3267]. Because the existence of one or more other genes for a polyamine transport protein on the vacuolar membrane was expected, we searched sequence databases for homologues of the protein encoded by TPO1. Membrane proteins encoded by the open reading frames YGR138c (TPO2), YPR156c (TPO3) and YOR273c (TPO4) were postulated to be polyamine transporters and, indeed, were subsequently shown to be polyamine transport proteins on the vacuolar membrane. Cells overexpressing these genes were resistant to polyamine toxicity and showed an increase in polyamine uptake activity and polyamine content in vacuoles. Furthermore, cells in which these genes were disrupted showed an increased sensitivity to polyamine toxicity and a decrease in polyamine uptake activity and polyamine content in vacuoles. Resistance to polyamine toxicity in cells overexpressing the genes was overcome by bafilomycin A(1), an inhibitor of the vacuolar H(+)-ATPase. Among the four polyamine transporters, those encoded by TPO2 and TPO3 were specific for spermine, whereas those encoded by TPO1 and TPO4 recognized spermidine and spermine. These results suggest that polyamine content in the cytoplasm of yeast is elaborately regulated by several polyamine transport systems in vacuoles. Furthermore, it was shown that Glu-207, Glu-324 (or Glu-323) and Glu-574 of TPO1 protein were important for the transport activity.

PotF protein is a periplasmic substrate-binding protein of the putrescine transport system in Escherichia coli. We have determined the crystal structure of PotF protein in complex with the substrate at 2.3-Å resolution. The PotF molecule has dimensions of 54 ؋ 42 ؋ 30 Å and consists of two similar globular domains. The PotF structure is reminiscent of other periplasmic receptors with a highest structural homology to another polyamine-binding protein, PotD. Putrescine is tightly bound in the deep cleft between the two domains of PotF through 12 hydrogen bonds and 36 van der Waals interactions. The comparison of the PotF structure with that of PotD provides the insight into the differences in the specificity between the two proteins. The PotF structure, in combination with the mutational analysis, revealed the residues crucial for putrescine binding (Trp-37, Ser-85, Glu-185, Trp-244, Asp-247, and Asp-278) and the importance of water molecules for putrescine recognition.