A novel homozygous mutation in GAD1 gene described in a schizophrenic patient impairs activity and dimerization of GAD67 enzyme

Recently, by whole exome sequencing of schizophrenia (SCZ) patients, we identified a subject that was homozygous for a novel missense substitution (c.391 A > G) in the glutamate acid decarboxylase 1 (GAD1) gene. GAD1 encodes for GAD67 enzyme, catalyzing the production of gamma-aminobutyric acid (GABA) from L-glutamic acid. Here, we studied the impact of this mutation on GAD67 activity, dimerization and subcellular localization. Biochemical assay revealed that c.391 A > G reduces GAD67 enzymatic activity by ~30%, probably due to the impaired homodimerization of homozygous mutants as highlighted by proximity ligation assays. The mutational screening of 120 genes of the "GABAergic system" in a cohort of 4,225 SCZ cases and 5,834 controls (dbGaP: phs000473.v1.p2), did not identify other cases that were homozygous for ultra-rare variants in GAD1, but highlighted an increased frequency of cases that were homozygous for rare variants in genes of the GABA system (SCZ: 0.14% vs. Controls: 0.00%; p-value = 0.0055). In conclusion, this study demonstrates the functional impact of c.391 A > G variant and its biological effect makes it a good candidate as risk variant for SCZ. This study also supports an involvement of ultra-rare variants in GABAergic genes in the etiopathogenesis of SCZ

Genomic and biochemical characterization of sialic acid acetylesterase (siae) in zebrafish

Sialic acid acetylesterase (SIAE) removes acetyl moieties from the carbon 9 and 4 hydroxyl groups of sialic acid and recently a debate has been opened on its association to autoimmunity. Trying to get new insights on this intriguing enzyme we have studied siae in zebrafish (Danio rerio). In this teleost siae encodes for a polypeptide with a high degree of sequence identity to human and mouse counterparts. Zebrafish Siae behavior upon transient expression in COS7 cells is comparable to human enzyme concerning pH optimum of enzyme activity, subcellular localization and glycosylation. In addition, and as already observed in case of human SIAE, the glycosylated form of the enzyme from zebrafish is released into the culture media. During embryogenesis, in situ hybridization experiments demonstrate that siae transcript is always detectable during development, with a more specific expression in the central nervous system, in pronephric ducts and liver in the more advanced stages of the embryo development. In adult fish an increasing amount of siae mRNA is detectable in heart, eye, muscle, liver, brain, kidney and ovary. These results provide novel information about Siae and point out zebrafish as animal model to better understand the biological role(s) of this rather puzzling enzyme in vertebrates, regarding immune system function and the development of central nervous system

Distribution of variants identified in NEU1.

<p>Schematic representation of NEU1 protein, with amino acid position on the X axis. Black vertical lines divide the 6 exons of <i>NEU1</i> gene. Line graph indicates the conservation score for each amino acid position as calculated by ScoreCons. The identified missense variants are represented as squares, with Y values corresponding to PolyPhen score. The missense variants already identified as pathological alleles in sialidosis are represented as diagonal crosses. Below the graph, schematic representation of NEU1 structural elements: full color blocks represent beta-strand elements, while crossed blocks represent helices. Color code identifies the 6 blades of the β-propeller structure as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104229#pone.0104229-Chavas1" target="_blank">[35]</a>. Gray boxes represent the Asp-box elements and essential catalytic residues are indicated with dots and their residue number. Positions of all elements are relative to the amino acid position in the graph above.</p

Distribution of SNVs in secondary structural elements of NEU1 protein.

<p>Numbers in brackets represent the p-value associated to the enrichment of SNVs in the corresponding category calculated as described in Methods. Classification of variants: mis, missense; syn, synonymous.</p

Sialidase activity assay and immunoblotting analysis.

<p>(A) Sialidase specific activity calculated for COS7 cells non transfected (NT), transfected with NEU1 wt alone (NEU1), PPCA alone (PPCA), NEU1 wt and PPCA (WT) and NEU1 mutants and PPCA. V217A and D234N mutant proteins resulted in a significant reduction in sialidase activity (p<0.05, marked with *). (B) Immunoblotting for PPCA and NEU1 proteins in each sample. NEU1 showed the expected signals between 40 and 46 kDa, corresponding to different glycosylation states; PPCA showed the expected signal at 32 kDa, corresponding to the heavy peptide of the active form of the protein. α-tub was used for protein loading normalization. The image is representative of 3 replicate experiments.</p

NEU1 structure analysis and localization of the 9 candidate missense mutations.

<p>Top (A) and lateral (B) views of the structural model of NEU1 protein. The two amino acids V217 and D234, identified in this work as affected by new putative pathological mutations, are shown in red; the 7 amino acids affected by the other candidate missense variants tested are shown in green. Other amino acids already reported as mutated in sialidosis and likely involved in NEU1-PPCA interaction <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104229#pone.0104229-Lukong1" target="_blank">[28]</a> are shown in blue. The black arrow in (B) indicates the side of the catalytic crevice. (C) Detailed view of the localization of V217 and D234. These two amino acids, together with G218, L231, W240, G243 and A298 define a region of the NEU1 protein that could represent an important site in the surface interaction with PPCA partner.</p

Summary information on <i>NEU1</i> gene and related SNVs.

<p><i>NEU1</i> cDNA sequence refers to NM_000434.3, protein sequence refers to NP_000425.1. LoF, loss of function; UTR, located in the untranslated regions. dN/dS measures the level of evolutionary constrain acting on the <i>NEU1</i> gene (see Methods).</p

Subcellular localization study of V217A and D234N NEU1 mutant proteins.

<p>Confocal microscopy images showing the subcellular distribution of NEU1 wild-type, V217A and D234N mutants. NEU1 proteins were detected using specific rabbit anti-NEU1 antibody and revealed with Alexa-555 secondary antibody. Wild-type NEU1 labeling resulted in a vesicular pattern (A), mainly colocalizing with the lysosomal marker LAMP1 detected with mouse anti-LAMP1 and revealed with Alexa-405 secondary antibody (B, merge in C). The V217A (D) and D234N (G) mutant proteins showed a tubulo-reticular localization. Almost no colocalization between V217A and D234N mutants and LAMP1 (E–F and H–I) could be detected. Insets represent enlargement of the indicated areas.</p

Distribution of SNVs in the exon of <i>NEU1</i> gene.

<p>Based on <i>NEU1</i> cDNA sequence NM_000434.3. Numbers in brackets represent the p-value associated to the enrichment of SNVs in the corresponding exon calculated as described in Methods. Classification of variants: mis, missense; syn, synonymous; UTR, located in the untranslated regions.</p