Involvement of ADGRV1 Gene in Familial Forms of Genetic Generalized Epilepsy.

Background: Genetic generalized epilepsies (GGE) including childhood absence epilepsy (CAE), juvenile absence epilepsy (JAE), juvenile myoclonic epilepsy (JME), and GGE with tonic-clonic seizures alone (GGE-TCS), are common types of epilepsy mostly determined by a polygenic mode of inheritance. Recent studies showed that susceptibility genes for GGE are numerous, and their variants rare, challenging their identification. In this study, we aimed to assess GGE genetic etiology in a Sudanese population. Methods: We performed whole-exome sequencing (WES) on DNA of 40 patients from 20 Sudanese families with GGE searching for candidate susceptibility variants, which were prioritized by CADD software and functional features of the corresponding gene. We assessed their segregation in 138 individuals and performed genotype-phenotype correlations. Results: In a family including three sibs with GGE-TCS, we identified a rare missense variant in ADGRV1 encoding an adhesion G protein-coupled receptor V1, which was already involved in the autosomal recessive Usher type C syndrome. In addition, five other ADGRV1 rare missense variants were identified in four additional families and absent from 119 Sudanese controls. In one of these families, an ADGRV1 variant was found at a homozygous state, in a female more severely affected than her heterozygous brother, suggesting a gene dosage effect. In the five families, GGE phenotype was statistically associated with ADGRV1 variants (0R = 0.9 103). Conclusion: This study highly supports, for the first time, the involvement of ADGRV1 missense variants in familial GGE and that ADGRV1 is a susceptibility gene for CAE/JAE and GGE-TCS phenotypes.

Clinical and genetic study of Tunisian families with genetic generalized epilepsy: contribution of CACNA1H and MAST4 genes.

Genetic generalized epilepsies (GGE) (childhood absence epilepsy (CAE), juvenile myoclonic epilepsy (JME) and epilepsy with generalized tonic-clonic seizures (GTCS)) are mainly determined by genetic factors. Since few mutations were identified in rare families with autosomal dominant GGE, a polygenic inheritance was suspected in most patients. Recent studies on large American or European cohorts of sporadic cases showed that susceptibility genes were numerous although their variants were rare, making their identification difficult. Here, we reported clinical and genetic characteristics of 30 Tunisian GGE families, including 71 GGE patients. The phenotype was close to that in sporadic cases. Nineteen pedigrees had a homogeneous type of GGE (JME-CAE-CGTS), and 11 combined these epileptic syndromes. Rare non-synonymous variants were selected in probands using a targeted panel of 30 candidate genes and their segregation was determined in families. Molecular studies incriminated different genes, mainly CACNA1H and MAST4. The segregation of at least two variants in different genes in some pedigrees was compatible with the hypothesis of an oligogenic inheritance, which was in accordance with the relatively low frequency of consanguineous probands. Since at least 2 susceptibility genes were likely shared by different populations, genetic factors involved in the majority of Tunisian GGE families remain to be discovered. Their identification should be easier in families with a homogeneous type of GGE, in which an intra-familial genetic homogeneity could be suspected.

Loss of Function of GALNT2 Lowers High-Density Lipoproteins in Humans, Nonhuman Primates, and Rodents.

Human genetics studies have implicated GALNT2, encoding GalNAc-T2, as a regulator of high-density lipoprotein cholesterol (HDL-C) metabolism, but the mechanisms relating GALNT2 to HDL-C remain unclear. We investigated the impact of homozygous GALNT2 deficiency on HDL-C in humans and mammalian models. We identified two humans homozygous for loss-of-function mutations in GALNT2 who demonstrated low HDL-C. We also found that GALNT2 loss of function in mice, rats, and nonhuman primates decreased HDL-C. O-glycoproteomics studies of a human GALNT2-deficient subject validated ANGPTL3 and ApoC-III as GalNAc-T2 targets. Additional glycoproteomics in rodents identified targets influencing HDL-C, including phospholipid transfer protein (PLTP). GALNT2 deficiency reduced plasma PLTP activity in humans and rodents, and in mice this was rescued by reconstitution of hepatic Galnt2. We also found that GALNT2 GWAS SNPs associated with reduced HDL-C also correlate with lower hepatic GALNT2 expression. These results posit GALNT2 as a direct modulator of HDL metabolism across mammals.

Role of the phosphoinositide phosphatase FIG4 gene in familial epilepsy with polymicrogyria.

OBJECTIVE: The aim of this study was to identify the causal gene in a consanguineous Moroccan family with temporo-occipital polymicrogyria, psychiatric manifestations, and epilepsy, previously mapped to the 6q16-q22 region. METHODS: We used exome sequencing and analyzed candidate variants in the 6q16-q22 locus, as well as a rescue assay in Fig4-null mouse fibroblasts and immunohistochemistry of Fig4-null mouse brains. RESULTS: A homozygous missense mutation (p.Asp783Val) in the phosphoinositide phosphatase gene FIG4 was identified. Pathogenicity of the variant was supported by impaired rescue of the enlarged vacuoles in transfected fibroblasts from Fig4-deficient mice. Histologic examination of Fig4-null mouse brain revealed neurodevelopmental impairment in the hippocampus, cortex, and cerebellum as well as impaired cerebellar gyration/foliation reminiscent of human cortical malformations. CONCLUSIONS: This study extends the spectrum of phenotypes associated with FIG4 mutations to include cortical malformation associated with seizures and psychiatric manifestations, in addition to the previously described Charcot-Marie-Tooth disease type 4J and Yunis-Varón syndrome.

Mutations of DEPDC5 cause autosomal dominant focal epilepsies.

The main familial focal epilepsies are autosomal dominant nocturnal frontal lobe epilepsy, familial temporal lobe epilepsy and familial focal epilepsy with variable foci. A frameshift mutation in the DEPDC5 gene (encoding DEP domain-containing protein 5) was identified in a family with focal epilepsy with variable foci by linkage analysis and exome sequencing. Subsequent pyrosequencing of DEPDC5 in a cohort of 15 additional families with focal epilepsies identified 4 nonsense mutations and 1 missense mutation. Our findings provided evidence of frequent (37%) loss-of-function mutations in DEPDC5 associated with a broad spectrum of focal epilepsies. The implication of a DEP (Dishevelled, Egl-10 and Pleckstrin) domain-containing protein that may be involved in membrane trafficking and/or G protein signaling opens new avenues for research.

Cyclotridecanone 2,4-dinitrophenylhydrazone.

In cyclotridecanone 2,4-dinitrophenylhydrazone, C(19)H(28)N(4)O(4), the 13-membered carbocycle exists in the triangular [337] conformation. The 2,4-dinitrophenylhydrazone group is almost perpendicular to the 13-membered ring, with a dihedral angle of 82.66 (2) degrees between the mean planes. The dinitrophenylhydrazone rings are packed parallel to each other and separated by 3.28 (1) A. The NH group forms an intramolecular hydrogen bond to a nitro O atom, and there is a weaker C-H...O interaction between a cyclotridecane CH group and a symmetry-related 4-nitro O atom, with a C...O distance of 3.436 (2) A and a 150 degrees angle about the H atom. The structure, in combination with additional evidence, indicates that [337] is the preferred conformation of cyclotridecane and other simple 13-membered rings.

Cyclopentadecanone 2,4-dinitrophenylhydrazone.

In the title compound, C(21)H(32)N(4)O(4), no disorder is present in the 15-membered hydrocarbon ring, which exists in an unsymmetrical quinquangular [12345] conformation. The 2,4-dinitrophenylhydrazone group is approximately perpendicular to the C(15) ring, with a dihedral angle of 84.66 (1) degrees between their best planes.

Cyclopentadecanone.

In the crystal structure, the title compound (exaltone), C(15)H(28)O, exhibits no disorder, and the 15-membered ring exists in the quinquangular [13353] C(1) symmetry conformation. The crystal exhibits nonmerohedral twinning by twofold rotation about [100], but adjustment of the temperature to 90 K causes the [101] distance to equal the c axial length, allowing the twinning to be treated as pseudo-merohedral. The literature description of the structure as disordered orthorhombic with halved a-axis length is corrected.

Dynamics of mutated GFAP aggregates revealed by real-time imaging of an astrocyte model of Alexander disease.

Alexander disease (AxD) is a rare neurodegenerative disorder characterized by large cytoplasmic aggregates in astrocytes and myelin abnormalities and caused by dominant mutations in the gene encoding glial fibrillary acidic protein (GFAP), the main intermediate filament protein in astrocytes. We tested the effects of three mutations (R236H, R76H and L232P) associated with AxD in cells transiently expressing mutated GFAP fused to green fluorescent protein (GFP). Mutated GFAP-GFP expressed in astrocytes formed networks or aggregates similar to those found in the brains of patients with the disease. Time-lapse recordings of living astrocytes showed that aggregates of mutated GFAP-GFP may either disappear, associated with cell survival, or coalesce in a huge juxtanuclear structure associated with cell death. Immunolabeling of fixed cells suggested that this gathering of aggregates forms an aggresome-like structure. Proteasome inhibition and immunoprecipitation assays revealed mutated GFAP-GFP ubiquitination, suggesting a role of the ubiquitin-proteasome system in the disaggregation process. In astrocytes from wild-type-, GFAP-, and vimentin-deficient mice, mutated GFAP-GFP aggregated or formed a network, depending on qualitative and quantitative interactions with normal intermediate filament partners. Particularly, vimentin displayed an anti-aggregation effect on mutated GFAP. Our data indicate a dynamic and reversible aggregation of mutated GFAP, suggesting that therapeutic approaches may be possible.

Conformational equilibria in formic acid and the adduct of formic acid and hexafluoroacetone, HCO2C(CF3)2OH.

Low-temperature 1H and 13C NMR spectra of formic acid (1) showed separate signals for the E and Z conformations in solvents containing a hydrogen bond acceptor, dimethyl ether. The population of E-1 (6.2% in 3:1:1 CHClF2/CHCl2F/(CH3)2O) was larger than that for 13C-labeled methyl formate in the same solvent (0.2%), which indicated that the relative populations are not determined by steric effects. The free-energy difference between the E and Z conformations of 1 was 0.9 kcal/mol. In a 1:3 CD2Cl2/(CH3)2O solvent mixture, peaks for E and Z conformations were found at low temperatures by 1H and 13C NMR for both formic acid and an adduct with hexafluoroacetone, HCO2C(CF3)2OH (2). The population of E-1 in this solvent mixture was 4.3% by 13C NMR. The carbon spectrum showed two peaks in the carbonyl carbon region of nearly equal intensities at -151.6 degrees C, with E-2 (48%) absorbing downfield of the major Z-2 (52%). The large population of E-2 confirms that electron-withdrawing groups R' in RCO2R' enhance the populations of the E-isomers. The free-energy barriers for 2 of 6.24 (E-to-Z) and 6.26 kcal/mol (Z-to-E) were determined from rate constants obtained by line shape analysis at -143.2 degrees C.

Interconversion of carbon sites in boat-chair-boat cyclodecane; conformations of chlorocyclodecane and cyclodecyl acetate.

Interconversion of carbon sites in boat-chair-boat (BCB) cyclodecane occurs by way of the twist-boat-chair (TBC) conformation, which predicts that C-1 exchanges with C-4, etc. The previously obtained low-temperature 13C spectra could be matched by assuming (1,4) or (1,3) exchange, but not (1,2) exchange. A free-energy barrier of 5.54 kcal/mol was obtained for conversion of BCB to TBC at -137.4 degrees C. The major conformation of chlorocyclodecane and cyclodecyl acetate is assigned to the IIIe BCB.

Cyclodecyl 4-nitrophenylacetate.

Cyclodecyl 4-nitrophenylacetate, C18H25NO4, has its ten-membered ring in the expected diamond-lattice boat-chair-boat [2323] conformation, with the substituent 4-nitrophenylacetoxy group in the BCB IIIe position. The ester unit has the expected Z conformation, with an O=C-O-C torsion angle of -0.3 (3) degrees, and the connection to the benzene ring is nearly perpendicular to the ester, with an O=C-C-C torsion angle of 85.5 (2) degrees. An intermolecular contact exists between the ester C atom and a nitro O atom, having a C...O distance of 2.909 (2) A.

Conformations of cycloundecane.

Cycloundecane (1) was shown to exist at -183.1 degrees C as a mixture of the [12323] (approximately 59%) and [335] (approximately 41%) conformations. Populations were determined from the (13)C NMR spectrum, and assignments were based on the (13)C spectra, calculated free energies and chemical shifts, and information from the literature, including X-ray studies of solid derivatives and calculated barriers.

Conformational study of cis-1,4-di-tert-butylcyclohexane by dynamic NMR spectroscopy and computational methods. Observation of chair and twist-boat conformations.

[reaction: see text] Low-temperature 13C NMR spectra of cis-1,4-di-tert-butylcyclohexane (1) showed signals for the twist-boat (1a) and chair (1b) conformations. 13C NMR signals were assigned to specific carbons based on the different populations, different symmetries (time-averaged C(2v) for 1a and time-averaged C(s) for 1b), and calculated chemical shifts (GIAO, HF/6-311+G*). In addition to slow ring inversion and interconversion of the chair and twist-boat conformations, slow rotation of the tert-butyl groups was found. Most of the expected 13C peaks were observed. Free-energy barriers of 6.83 and 6.35 kcal/mol were found for interconversion of 1a (major) and 1b (minor) at -148.1 degrees C. Conformational space was searched with Allinger's MM3 and MM4 programs, and free energies were obtained for several low-energy conformations 1a-c. Calculations were repeated with ab initio methods up to the HF/6-311+G* level. Molecular symmetries, relative free energies, relative enthalpies and entropies, frequencies, and NMR chemical shifts were obtained. A boat conformation (1d; C(2v) symmetry) was generated and optimized as a transition state by ab initio, MM3, and MM4 calculations.

Conformational study of 1,2-cycloundecadiene by dynamic NMR spectroscopy and computational methods.

Solutions of 1,2-cycloundecadiene in propane were studied by low-temperature (13)C NMR spectroscopy. A total of 17 peaks were observed at -166.7 degrees C, corresponding to two conformations of similar populations, one of C(1) symmetry (11 peaks) and the other of C(2) symmetry. The line shapes show that the predominant pathway for exchange of the topomers (C(1) and C(1)') of the C(1) conformation does not include the C(2) conformation. From the (13)C spectra, free-energy barriers of 8.38 +/- 0.15, 9.45 +/- 0.15, and 9.35 +/- 0.15 kcal/mol were determined for the C(1) to C(1)', (C(1) + C(1)') to C(2), and C(2) to (C(1) + C(1)') conversions, respectively, at -72.2 degrees C. The NMR results for this compound are discussed in terms of the conformations predicted by molecular mechanics calculations obtained with Allinger's MM3 program. Ab initio calculations of free energies are also reported at the HF/ 6-311G level for 25 conformations.

Conformational Study of trans-Cyclododecene by Dynamic NMR Spectroscopy and Computational Methods.

The (13)C spectrum of trans-cyclododecene (1) dissolved in propane showed seven peaks for the olefinic carbons at -164.5 degrees C, corresponding to three conformations of C(1) symmetry and a fourth conformation, with a population of 20.1%, of C(2) symmetry. The populations of the C(1) conformations were 57.0, 18.6, and 4.3%. Conformational space was searched for 1 using MM2. Free energies and populations were calculated by MM3, and the results of the low-temperature NMR study are discussed in terms of these calculations and the (13)C chemical shifts calculated for eight conformations by the GIAO method at the HF/6-311G level. The conformations of 1 and cyclododecane (2) are compared.

Conformational Study of Oxacyclooctan-2-one by Dynamic NMR Spectroscopy and Computational Methods.

The slow-exchange (13)C NMR spectrum of the eight-membered lactone, oxacyclooctan-2-one (1), at -154.2 degrees C shows the presence of two conformations with populations of 25.6 and 74.4%. Free-energy barriers of 6.98 and 7.23 kcal/mol were determined at the coalescence temperature for the carbonyl carbon peak (-128.6 degrees C). The (1)H NMR spectrum of the CH(2)O protons splits into two multiplets of equal intensity by -100 degrees C, and the corresponding free-energy barrier of 9.16 kcal/mol at -85.2 degrees C was suggested to be associated with exchange of the geminal protons. Relative free energies and populations at +25 and -160 degrees C were estimated by using Allinger's MM3 program, and free energies and dipole moments were obtained for three conformations using ab initio calculations at the HF/6-311G and MP2/6-311G levels. The spectra were interpreted in terms of two conformations having the E configuration of the lactone group and resembling the boat-chair conformation of cyclooctane.