Etiological involvement of KCND1 variants in an X-linked neurodevelopmental disorder with variable expressivity

Utilizing trio whole-exome sequencing and a gene matching approach, we identified a cohort of 18 male individuals from 17 families with hemizygous variants in KCND1, including two de novo missense variants, three maternally inherited protein-truncating variants, and 12 maternally inherited missense variants. Affected subjects present with a neurodevelopmental disorder characterized by diverse neurological abnormalities, mostly delays in different developmental domains, but also distinct neuropsychiatric signs and epilepsy. Heterozygous carrier mothers are clinically unaffected. KCND1 encodes the α-subunit of Kv4.1 voltage-gated potassium channels. All variant-associated amino acid substitutions affect either the cytoplasmic N- or C-terminus of the channel protein except for two occurring in transmembrane segments 1 and 4. Kv4.1 channels were functionally characterized in the absence and presence of auxiliary β subunits. Variant-specific alterations of biophysical channel properties were diverse and varied in magnitude. Genetic data analysis in combination with our functional assessment shows that Kv4.1 channel dysfunction is involved in the pathogenesis of an X-linked neurodevelopmental disorder frequently associated with a variable neuropsychiatric clinical phenotype.</p

Bi-allelic ACBD6 variants lead to a neurodevelopmental syndrome with progressive and complex movement disorders

The acyl-CoA-binding domain-containing protein 6 (ACBD6) is ubiquitously expressed, plays a role in the acylation of lipids and proteins, and regulates the N-myristoylation of proteins via N-myristoyltransferase enzymes (NMTs). However, its precise function in cells is still unclear, as is the consequence of ACBD6 defects on human pathophysiology. Utilizing exome sequencing and extensive international data sharing efforts, we identified 45 affected individuals from 28 unrelated families (consanguinity 93%) with bi-allelic pathogenic, predominantly loss-of-function (18/20) variants in ACBD6. We generated zebrafish and Xenopus tropicalis acbd6 knockouts by CRISPR/Cas9 and characterized the role of ACBD6 on protein N-myristoylation with YnMyr chemical proteomics in the model organisms and human cells, with the latter also being subjected further to ACBD6 peroxisomal localization studies. The affected individuals (23 males and 22 females), with ages ranging from 1 to 50 years old, typically present with a complex and progressive disease involving moderate-to-severe global developmental delay/intellectual disability (100%) with significant expressive language impairment (98%), movement disorders (97%), facial dysmorphism (95%), and mild cerebellar ataxia (85%) associated with gait impairment (94%), limb spasticity/hypertonia (76%), oculomotor (71%) and behavioural abnormalities (65%), overweight (59%), microcephaly (39%) and epilepsy (33%). The most conspicuous and common movement disorder was dystonia (94%), frequently leading to early-onset progressive postural deformities (97%), limb dystonia (55%), and cervical dystonia (31%). A jerky tremor in the upper limbs (63%), a mild head tremor (59%), parkinsonism/hypokinesia developing with advancing age (32%), and simple motor and vocal tics were among other frequent movement disorders. Midline brain malformations including corpus callosum abnormalities (70%), hypoplasia/agenesis of the anterior commissure (66%), short midbrain and small inferior cerebellar vermis (38% each), as well as hypertrophy of the clava (24%) were common neuroimaging findings. acbd6-deficient zebrafish and Xenopus models effectively recapitulated many clinical phenotypes reported in patients including movement disorders, progressive neuromotor impairment, seizures, microcephaly, craniofacial dysmorphism, and midbrain defects accompanied by developmental delay with increased mortality over time. Unlike ACBD5, ACBD6 did not show a peroxisomal localisation and ACBD6-deficiency was not associated with altered peroxisomal parameters in patient fibroblasts. Significant differences in YnMyr-labelling were observed for 68 co- and 18 post-translationally N-myristoylated proteins in patient-derived fibroblasts. N-Myristoylation was similarly affected in acbd6-deficient zebrafish and Xenopus tropicalis models, including Fus, Marcks, and Chchd-related proteins implicated in neurological diseases. The present study provides evidence that bi-allelic pathogenic variants in ACBD6 lead to a distinct neurodevelopmental syndrome accompanied by complex and progressive cognitive and movement disorders

Das Mikrodeletionssyndrom 20q11–q12

&lt;jats:title&gt;Zusammenfassung&lt;/jats:title&gt;&lt;jats:p&gt;Dieser Artikel beschreibt das Spektrum des seltenen, aber rekurrenten Mikrodeletionssyndrom 20q11–q12 anhand der Fallbeschreibung eines Jungen mit charakteristischem Phänotyp. Der Patient weist eine milde kombinierte Entwicklungsverzögerung, einen Kleinwuchs, Mikrozephalie, milde Ptosis, Knick-Senk-Füße und eine sehr helle (nichtfamiliäre) Komplexion (Haut, Haare, Iris) mit milden fazialen Auffälligkeiten auf. Eine Trio-Exom-Analyse identifizierte eine De-novo-Mikrodeletion in 20q11.23–q12. Retrospektiv entspricht insbesondere der faziale Phänotyp exakt dem vormals beschriebenen; der Fall hätte ggf. mittels syndromologischer Verdachtsdiagnose und Array-Analyse gelöst werden können.&lt;/jats:p&gt

αPIX Is a Trafficking Regulator that Balances Recycling and Degradation of the Epidermal Growth Factor Receptor.

Endosomal sorting is an essential control mechanism for signaling through the epidermal growth factor receptor (EGFR). We report here that the guanine nucleotide exchange factor αPIX, which modulates the activity of Rho-GTPases, is a potent bimodal regulator of EGFR trafficking. αPIX interacts with the E3 ubiquitin ligase c-Cbl, an enzyme that attaches ubiquitin to EGFR, thereby labelling this tyrosine kinase receptor for lysosomal degradation. We show that EGF stimulation induces αPIX::c-Cbl complex formation. Simultaneously, αPIX and c-Cbl protein levels decrease, which depends on both αPIX binding to c-Cbl and c-Cbl ubiquitin ligase activity. Through interaction αPIX sequesters c-Cbl from EGFR and this results in reduced EGFR ubiquitination and decreased EGFR degradation upon EGF treatment. However, quantitatively more decisive for cellular EGFR distribution than impaired EGFR degradation is a strong stimulating effect of αPIX on EGFR recycling to the cell surface. This function depends on the GIT binding domain of αPIX but not on interaction with c-Cbl or αPIX exchange activity. In summary, our data demonstrate a previously unappreciated function of αPIX as a strong promoter of EGFR recycling. We suggest that the novel recycling regulator αPIX and the degradation factor c-Cbl closely cooperate in the regulation of EGFR trafficking: uncomplexed αPIX and c-Cbl mediate a positive and a negative feedback on EGFR signaling, respectively; αPIX::c-Cbl complex formation, however, results in mutual inhibition, which may reflect a stable condition in the homeostasis of EGF-induced signal flow

αPIX::c-Cbl complex formation and degradation.

<p>A. EGF regulates complex formation of αPIX and c-Cbl. COS-7 cells transiently co-expressing HA-αPIX^WT and c-Cbl^WT were serum-starved or cultured under basal growth conditions (+S). Starved cells were stimulated with 5 ng/ml EGF for 5, 10, 30 or 60 min at 37°C (t_EGF) or left untreated (0 min). αPIX was immunoprecipitated from cell extracts by using anti-HA antibodies and protein levels of HA-αPIX, c-Cbl and GAPDH were determined in cell lysates (tcl) and precipitates (IP) by immunoblotting. Based on densitometric quantification of autoradiographic signals derived from immunoblots, the graphs show relative amounts of c-Cbl co-precipitated with HA-αPIX in unstimulated cells and at 30 min upon EGF induction. Amounts of c-Cbl in the precipitates were normalized to total c-Cbl and considered as 1 for unstimulated cells (0 min). Data represent the mean of six (n = 6) independent experiments ± sd. <i>P</i> value was calculated by paired Student’s t-test. B. Downregulation of αPIX and c-Cbl depends on complex formation of these proteins. COS-7 cells transiently expressing various HA-αPIX and c-Cbl protein variants were cultivated under basal growth conditions (+S), serum starved (0 min) or serum-starved and stimulated with 5 ng/ml EGF for 15 or 30 min. Cells were harvested and protein levels of HA-αPIX, c-Cbl, and GAPDH were determined by immunoblotting. Based on densitometric quantification of autoradiographic signals derived from immunoblots, the graphs show relative amounts of HA-αPIX^WT and c-Cbl^WT in the total lysates from cells overexpressing HA-αPIX^WT and c-Cbl^WT. Measurements were normalized to GAPDH and considered as 1 for unstimulated cells (0 min t_EGF). Data represent the mean of five (n = 5) independent experiments ± sd. <i>P</i> values were calculated by paired Student’s t-test. C. Both proteasomal and lysosomal inhibitors prevent EGF-induced αPIX and c-Cbl degradation. Serum-starved COS-7 cells transiently co-expressing HA-αPIX^WT and c-Cbl^WT were incubated with 20 μM MG132 or 50 μM chloroquine for 6h or left untreated (vehicle). Upon stimulation with 25 ng/ml EGF for the indicated times, cell extracts were subjected to SDS-PAGE and immunoblotting using anti-HA and anti-Cbl antibodies. Blots were reprobed with anti-GAPDH antibody to test for loading equality. Based on densitometric quantification of autoradiographic signals derived from immunoblots, the graphs show relative amounts of HA-αPIX and Cbl in the cell lysates. Measurements were normalized to GAPDH and considered as 1 for unstimulated cells (0 min t_EGF). Data represent the mean of four (n = 4) independent experiments ± sd. <i>P</i> values were calculated by unpaired Student’s t-test.</p

αPIX binds to the E3 ubiquitin ligase c-Cbl.

<p>A. Lysates from CHO-K1 cells were subjected to co-immunoprecipitation, either using rabbit IgG or rabbit anti-Cbl (S.C., Santa Cruz) antibodies. Total cell lysates (tcl) and immunoprecipitates (p) were resolved on an SDS-polyacrylamide gel and analyzed by immunoblotting using the indicated antibodies. B. Modular architecture of αPIX and c-Cbl. The protein domains of αPIX (CH, calponin homology; SH3, src-homology 3; DH, Dbl homology; PH, pleckstrin homology; GBD, GIT-binding domain; CC, coiled-coil domain) and c-Cbl (TKB, tyrosine-kinase-binding region; RING, RING finger domain; PRD, proline-rich domain; PKPFPR binding motif; UBA, ubiquitin associated domain) are schematically shown. Amino acid substitutions that are functionally important for this study are indicated. The total number of amino acids (aa) for αPIX and c-Cbl is given. C. Trp¹⁹⁷ in αPIX and Arg⁸²⁹ in c-Cbl are essential for the αPIX::c-Cbl interaction. COS-7 cells were transfected with the indicated expression constructs. HA-tagged αPIX was immunoprecipitated from cell extracts by using anti-HA-conjugated agarose beads. After SDS-PAGE and western blotting, immunoprecipitates (IP) and total cell lysates (tcl) were probed with anti-HA and anti-Cbl antibodies. The HA-membrane was re-probed using anti-GAPDH antibodies to control for equal loading.</p

Model depicting the balancing effect of αPIX on EGFR trafficking to maintain EGFR signaling homeostasis.

<p>(For details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132737#sec010" target="_blank">discussion</a>.)</p

Stimulation of EGFR recycling is the dominant αPIX function during EGFR trafficking.

<p>A. CHO cells stably expressing αPIX^WT or CAT (control) were transfected with EGFR expression constructs. Following serum starvation overnight, surface proteins were biotinylated on ice and cells were stimulated with 25 ng/ml EGF for 15, 30 or 60 min at 37°C to induce EGF receptor trafficking. A parallel culture was left unstimulated (0 min). Cells were transferred to 4°C, surface proteins were de-biotinylated and intracellular biotinylated proteins were precipitated from cell extracts. Representative autoradiographs show EGFR levels in total cell lysates (tcl) and precipitates (p) upon SDS-PAGE and immunoblotting. GAPDH served as a loading control. Based on densitometric quantification of autoradiographic signals the graphs show relative amounts of intracellular EGFR. Amounts of precipitated EGFR were normalized to total EGFR levels and considered as 100% in control cells after 60 min EGF stimulation (note: standard deviation for control cells at 60 min t_EGF was calculated subsequent to normalization to total EGFR levels). Data represent the mean of three independent experiments ± sd. For <i>P</i> value was calculated by paired Student’s t-test. B. Immunocytochemical analysis of EGFR distribution. Stable αPIX^WT and control (CAT) CHO cells were transfected with EGFR constructs and serum-starved overnight. Cells were stimulated with 25 ng/ml EGF for 15 or 60 min at 37°C to induce EGF receptor trafficking. After fixation, EGFR was visualized by anti-EGFR antibodies followed by Alexa Fluor488-conjugated antibodies and the nucleus was detected by staining with DAPI. Note the enrichment of EGFR at the plasma membrane in αPIX^WT overexpressing cells upon 60 min EGF stimulation (arrowheads, lower panel). 25 cells each [stably expressing CAT (control) and αPIX^WT cells] derived from three independent experiments have been analyzed, representative cells are shown. Scale bars, 20 μm. C. Serum-starved COS-7 cells transiently expressing HA-αPIX^WT were stimulated with 25 ng/ml Alexa Fluor 488-conjugated EGF (EGF488) for 15 or 60 min. Subsequently, extracellular receptor-bound EGF was removed and cells were fixed. HA-tagged αPIX was visualized by staining with anti-HA antibodies followed by Alexa Flour 546-conjugated secondary antibodies and the nucleus was detected by staining with DAPI. Dotted lines indicate αPIX-expressing cells, dashed lines indicate untransfected cells. 50 cells each (non-transfected cells and HA-αPIX^WT overexpressing cells) derived from three independent specimen have been examined, representative cells are shown. Scale bars, 20 μm.</p

αPIX interferes with EGFR ubiquitination and degradation.

<p>A. CHO cells stably expressing the indicated αPIX protein variants or CAT (control) were transfected with an EGFR expression construct. The experimental procedure was essentially the same as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132737#pone.0132737.g003" target="_blank">Fig 3A</a>, except for the medium that was supplemented with 0.3 mM of the recycling inhibitor primaquine to block EGFR recycling. Cells were harvested after various times of rewarming followed by surface de-biotinylation (5, 15, 30 min) or before rewarming (0 min). Intracellular biotinylated proteins were precipitated from cell extracts and cell lysates (tcl) and precipitates (p) were subjected to immunoblotting using anti-EGFR antibodies. B. Graphs show relative amounts of intracellular EGFR derived from densitometric quantification of autoradiographic signals obtained from EGFR degradation assays (A). Precipitated (intracellular) EGFR fractions were normalized to total EGFR levels and considered as 100% in cultures that haven’t been rewarmed (0 min). Data represent the mean of three (n = 3) independent experiments ± sd. <i>P</i> values were calculated by unpaired Student’s t-test. C. c-Cbl co-expression rescues αPIX^WT-induced inhibition of EGFR degradation. CHO cells stably expressing αPIX^WT were co-transfected with EGFR and c-Cbl expression constructs and subsequently treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132737#pone.0132737.g003" target="_blank">Fig 3A</a>. After immunoblotting cell lysates (tcl) were probed with anti c-Cbl and anti-EGFR antibodies, and precipitates (p) were probed with anti-EGFR antibodies. D. αPIX^WT sequesters c-Cbl from EGF receptors. COS-7 cells were transfected with expression constructs as indicated. Endogenous EGFR was immunoprecipitated from cell extracts by using anti-EGFR antibodies. Upon SDS-PAGE and western blotting, precipitates (IP) and total cell lysates (tcl) were probed with anti-EGFR and anti-Cbl antibodies. Expression of αPIX^WT was demonstrated by immunodetection with anti-HA antibodies. E. αPIX^WT reduces c-Cbl-mediated EGFR ubiquitination. COS-7 cells were transiently co-transfected with <i>c-Cbl</i> and FLAG-tagged <i>αPIX</i> expression constructs (as indicated) together with HA-tagged ubiquitin and EGFR expression constructs. For control purpose cells were transfected with empty FLAG-vector. Subsequent to incubation under serum-free culture conditions overnight, cells were stimulated with 20 ng/ml EGF for 30 min and harvested. EGFR was immunoprecipitated with anti-EGFR antibodies and protein A-agarose and samples were subjected to SDS-PAGE and immunoblotting. Levels of ubiquitinated EGFR in precipitates (IP) were monitored by using anti-HA antibodies. EGFR levels in total cell lysates (tcl) and precipitates (IP) were determined by using anti-EGFR antibodies and expression of c-Cbl and FLAG-αPIX protein variants in total cell lysates was shown by using anti-c-Cbl and anti-FLAG antibodies, respectively. Tubulin served as a loading control. Representative blots from one out of three independent experiments are shown. Based on densitometric quantification of autoradiographic signals derived from immunoblots, the graph shows relative amounts of ubiquitinated EGFR. Amounts of HA-ubiquitinated EGFR in the precipitates were normalized to total EGFR and considered as 1 for cells expressing FLAG-αPIX^WT. Data represent the mean of three (n = 3) independent experiments ± sd. <i>P</i> value was calculated by paired Student’s t-test.</p

αPIX regulates EGFR trafficking.

<p>A. Stable αPIX^WT and control (CAT) CHO cell lines were transiently transfected with EGFR expression constructs. Following serum starvation, surface proteins were biotinylated and cells were stimulated with 25 ng/ml EGF for 30 min at 37°C (pulse) to induce EGF receptor trafficking. Then, cells were transferred to 4°C, residual surface biotin was removed and cells were rewarmed to 37°C for the indicated times (chase). Recycled surface proteins were de-biotinylated and intracellular biotinylated proteins were precipitated from cell extracts. Parallel cultures were harvested before rewarming (0 min). Representative autoradiographs show EGFR levels in precipitates (p) and total cell lysates (tcl). Equal loading was verified by reprobing membranes with anti-GAPDH antibodies. B. Based on densitometric quantification of autoradiographic signals derived from EGFR trafficking assays (A), the graphs show relative amounts of intracellular EGFR. Precipitated (intracellular) EGFR fractions were normalized to total EGFR levels and considered as 100% in cultures that haven’t been rewarmed (0 min). Data represent the mean of four (n = 4) independent experiments ± sd. <i>P</i> values were calculated by unpaired Student’s t-test. C. Serum-starved COS-7 cells transiently expressing HA-αPIX^WT were stimulated with 25 ng/ml EGF for 30 min (pulse). Subsequently, cells were either immediately fixed (0 min) or incubated in starvation medium for further 30 min (chase) and then fixed. HA-tagged αPIX was visualized by staining with anti-HA followed by Alexa Fluor 488-conjugated secondary antibodies. The early endosomal compartment was visualized by anti-EEA1 antibodies followed by Alexa Fluor546-conjugated antibodies and the nucleus was detected by staining with DAPI. Note the enlarged morphology (arrowheads, upper panel) and the reduced number (outlined cell, lower panel) of EEA1-positive vesicles in αPIX overexpressing cells compared to surrounding non-transfected cells (asterisks) at 0 min and 30 min chase, respectively. Specific details are shown enlarged at the right hand side of the images. 50 cells each (non-transfected cells and HA-αPIX^WT overexpressing cells) derived from three independent specimen have been examined, representative cells are shown. Scale bar, 20 μm.</p