The genetic landscape of autism spectrum disorder in the Middle Eastern population

Introduction: Autism spectrum disorder (ASD) is characterized by aberrations in social interaction and communication associated with repetitive behaviors and interests, with strong clinical heterogeneity. Genetic factors play an important role in ASD, but about 75% of ASD cases have an undetermined genetic risk.Methods: We extensively investigated an ASD cohort made of 102 families from the Middle Eastern population of Qatar. First, we investigated the copy number variations (CNV) contribution using genome-wide SNP arrays. Next, we employed Next Generation Sequencing (NGS) to identify de novo or inherited variants contributing to the ASD etiology and its associated comorbid conditions in families with complete trios (affected child and the parents).Results: Our analysis revealed 16 CNV regions located in genomic regions implicated in ASD. The analysis of the 88 ASD cases identified 41 genes in 39 ASD subjects with de novo (n = 24) or inherited variants (n = 22). We identified three novel de novo variants in new candidate genes for ASD (DTX4, ARMC6, and B3GNT3). Also, we have identified 15 de novo variants in genes that were previously implicated in ASD or related neurodevelopmental disorders (PHF21A, WASF1, TCF20, DEAF1, MED13, CREBBP, KDM6B,SMURF1, ADNP, CACNA1G, MYT1L, KIF13B, GRIA2, CHM, and KCNK9). Additionally, we defined eight novel recessive variants (RYR2, DNAH3, TSPYL2, UPF3B KDM5C, LYST, and WNK3), four of which were X-linked.Conclusion: Despite the ASD multifactorial etiology that hinders ASD genetic risk discovery, the number of identified novel or known putative ASD genetic variants was appreciable. Nevertheless, this study represents the first comprehensive characterization of ASD genetic risk in Qatar's Middle Eastern population

AKT1 interacts specifically with DNAJB3.

(a) SDS-PAGE analysis of the recombinant GST-DNAJB3 and GST control proteins expressed and purified from E. coli. Different fractions of the elutes were resolved by SDS-PAGE and stained with Coomassie blue. (b) GST-pull down experiments were carried out using GST-DNAJB3 recombinant protein or GST (a negative control) and whole cell lysate prepared from C2C12 cells. After binding, the resin was washed extensively with the binding buffer containing increasing concentrations (0.1–1 M) of NaCl. Bound proteins were eluted with 20 mM GSH followed by 1% SDS, resolved by SDS-PAGE and stained with silver nitrate. Proteins present in the 1 M NaCl wash were loaded in parallel. The binding of GST and GST-DNAJB3 without lysate was also included. The protein bands that interact specifically with GST-DNAJB3 are indicated by a red dot. As similar pattern was also obeseved in 3T3-L1 adipocytes and HepG2 cells (S1 Fig in S1 File). Splice points are shown as vertical arrows.</p

Relevance of AKT in DNAJB3-mediated ER stress.

Silencing the expression of AKT abolishes the protective effect of DNAJB3 on tunicamycin-induced ER stress in C2C12 cells. DNAJB3 fails to protect siRNA AKT-transfected C2C12 cells from tunicamycin-induced mRNA expression of ER stress marker (GRP78). All assays were performed at least in triplicate and a minimum of three independent experiments. Results are presented as means ± SEM and were plotted using GraphPad (Prism v7, La Jolla, CA). Shapiro-Wilk test was first performed for the normality test followed by parametric tests. We used unpaired t-tests or two-way analysis of variance to test gene (Scrambled- vs SiRNA) by treatment condition (vehicle vs tunicamycin) effects and one-way analysis of variance for comparison of the groups with post-hoc Tukey’s test for pairwise comparisons. A P-value <0.05 was considered statistically significant.</p

Structural details of DNAJB3 and AKT1.

(a) Modular organization of human DNAJB3 protein. DNAJB3 contains the canonical J domain near N-terminal and a long C-terminal portion. While J domain structure is known, the C-terminal structure is unknow. (b) Modular organization of AKT1 protein. AKT1 is comprised of three well known domains, designated Pleckstrin homology (PH) domain located at N-terminal, Protein kinase domain in the middle and Protein kinase_C domain, located at C-terminal. (c) Cartoon representation of the overall modelled human DNAJB3 (hDNAJB3) structure. Secondary structures are shown in salmon for α-helix, and purple for loops. (d) Cartoon representation of the overall modelled mouse DNAJB3 (mDNAJB3) structure. Secondary structures are shown in salmon for α-helix, sea green for β-strands, and purple for loops. (e) Structural alignment of modelled hDnajb3 (orange), mDNAJB3 (sea green) and NMR structure of the DNAJB6b (PDBID: 6U3R, grey). The structures are highly similar in overall fold with protrusion of C-terminal as CTD in case of mDNAJB3.</p

ITC analysis of DNAJB3: AKT1 interaction.

(a) ITC titration curve (upper panel) and binding isotherms (low panel) of DNAJB3: AKT1 interactions. The upper panels show the raw ITC data expressed as change in thermal power with respect to time over the period of titration. In the lower panels, change in molar heat is expressed as a function of molar ratio of titrant. The solid line in the lower panels indicates the non-linear least squares fit for the integrated data to a one-site binding model using the integrated ORIGIN software. The solid line in the lower panels indicates the non-linear least squares fit for the integrated data to a one-site binding model using the integrated ORIGIN software. (b) ITC measurements showing of titration curve (upper panel) and binding isotherm of DNAJB3 titration into Sox2-HMG protein. No binding was observed.</p

Contains all the supporting information and figures.

Contains all the supporting information and figures.</p

Docking of modelled DNAJB3 onto AKT1 and structural comparison.

(a) Molecular docking of modelled DNAJB3 (J domain: red, c-terminal: sea green) onto AKT1 (PDBID:3CQW, purple) represented as cartoon (left panel) and surface (right panel). (b) Structure of Dnaj-PKACα (PDBID: 4WB7) drawn in carton (left panel) and surface (right panel) representations. The PKACα is coloured golden and J domain as red. (c) Structure alignment of docked (DNAJB3: AKT1) structure to Dnaj-PKACα (PDBID: 4WB7).</p

S1 File -

Metabolic stress involved in several dysregulation disorders such as type 2 diabetes mellitus (T2DM) results in down regulation of several heat shock proteins (HSPs) including DNAJB3. This down regulation of HSPs is associated with insulin resistance (IR) and interventions which induce the heat shock response (HSR) help to increase the insulin sensitivity. Metabolic stress leads to changes in signaling pathways through increased activation of both c-jun N-terminal kinase-1 (JNK1) and the inhibitor of κB inflammatory kinase (IKKβ) which in turn leads to inactivation of insulin receptor substrates 1 and 2 (IRS-1 and IRS-2). DNAJB3 interacts with both JNK1 and IKKβ kinases to mitigate metabolic stress. In addition DNAJB3 also activates the PI3K-PKB/AKT pathway through increased phosphorylation of AKT1 and its substrate AS160, a Rab GTPase-activating protein, which results in mobilization of GLUT4 transporter protein and improved glucose uptake. We show through pull down that AK T1 is an interacting partner of DNAJB3, further confirmed by isothermal titration calorimetry (ITC) which quantified the avidity of AKT1 for DNAJB3. The binding interface was identified by combining protein modelling with docking of the AKT1-DNAJB3 complex. DNAJB3 is localized in the cytoplasm and ER, where it interacts directly with AKT1 and mobilizes AS160 for glucose transport. Inhibition of AKT1 resulted in loss of GLUT4 translocation activity mediated by DNAJB3 and also abolished the protective effect of DNAJB3 on tunicamycin-induced ER stress. Taken together, our findings provide evidence for a direct protein-protein interaction between DNAJB3 and AKT1 upon which DNAJB3 alleviates ER stress and promotes GLUT4 translocation.</div

DNAJB3 sub-cellular localization with ER marker using confocal microscopy.

Staining with anti-DNAJB3 in C2C12 cells reveals its non-nuclear localization (upper panel). C2C12 cells were transfected with DNAJB3 tagged with GFP (DNAJB3-GFP) and stained with anti-GRP78 (an ER marker) and found that they colocalize (lower panel). DAPI was used as control for nuclear staining.</p

Primer list and sequences.

Metabolic stress involved in several dysregulation disorders such as type 2 diabetes mellitus (T2DM) results in down regulation of several heat shock proteins (HSPs) including DNAJB3. This down regulation of HSPs is associated with insulin resistance (IR) and interventions which induce the heat shock response (HSR) help to increase the insulin sensitivity. Metabolic stress leads to changes in signaling pathways through increased activation of both c-jun N-terminal kinase-1 (JNK1) and the inhibitor of κB inflammatory kinase (IKKβ) which in turn leads to inactivation of insulin receptor substrates 1 and 2 (IRS-1 and IRS-2). DNAJB3 interacts with both JNK1 and IKKβ kinases to mitigate metabolic stress. In addition DNAJB3 also activates the PI3K-PKB/AKT pathway through increased phosphorylation of AKT1 and its substrate AS160, a Rab GTPase-activating protein, which results in mobilization of GLUT4 transporter protein and improved glucose uptake. We show through pull down that AK T1 is an interacting partner of DNAJB3, further confirmed by isothermal titration calorimetry (ITC) which quantified the avidity of AKT1 for DNAJB3. The binding interface was identified by combining protein modelling with docking of the AKT1-DNAJB3 complex. DNAJB3 is localized in the cytoplasm and ER, where it interacts directly with AKT1 and mobilizes AS160 for glucose transport. Inhibition of AKT1 resulted in loss of GLUT4 translocation activity mediated by DNAJB3 and also abolished the protective effect of DNAJB3 on tunicamycin-induced ER stress. Taken together, our findings provide evidence for a direct protein-protein interaction between DNAJB3 and AKT1 upon which DNAJB3 alleviates ER stress and promotes GLUT4 translocation.</div