pmh sfb dyrk1a  (Addgene inc)

Journal: Genetics in medicine : official journal of the American College of Medical Genetics

Article Title: Integrative approach to interpret DYRK1A variants, leading to a frequent neurodevelopmental disorder.

doi: 10.1038/s41436-021-01263-1

Figure Lengend Snippet: Fig. 1 Clinical score for Intellectual Disability associated to DYRK1A haploinsufficiency. (a) Clinical score out of 20 points established according to the most recurrent clinical features presented by patients (the weight assigned to each symptom being based on its recurrence): clinical symptoms are out of 15 points, while the facial appearance is out of 5 points. CA cerebral atrophy, CCA/H corpus callosum agenesis or hypoplasia, CeA cerebellar atrophy, EV enlarged ventricules. (b) Clinical scores calculated for individuals carrying pathogenic variants in DYRK1A reported here and for whom photographs were available (n = 21) (initial cohort, DYRK1A_I, scores 13–17.5 with a mean of 15.5), the previously published individuals (replication cohort, DYRK1A_R, scores 13.5–18.5, mean = 15.3) and the individuals affected with other frequent monogenic forms of intellectual disability (ID), associated to variants in ANKRD11, MED13L, DDX3X, ARID1B, SHANK3, TCF4, or KMT2A (scores 3–12.5, mean = 7). The clinical score for the individuals carrying missense or distal frameshift variants are indicated in yellow (test); the threshold of CSDYRK1A ≥13 appeared to be discriminant between individuals with loss-of-function (LoF) variants in DYRK1A (all ≥13) and individuals suffering from another form of ID (all < 13). A score above this threshold was therefore considered highly suggestive. We classified individuals with CSDYRK1A < 10 as poorly evocative and individuals with a CSDYRK1A between 10 and 13 as intermediate. Brown–Forsythe and Welsh analysis of variance (ANOVA) tests with Dunnett’s T 3 multiple comparisons test were performed. ns not significant; **p < 0.01; ***

Article Snippet: In vitro analysis of variant effect on DYRK1A protein DYRK1A expression plasmids (addgene #101770, Huen lab) were generated from the pMH-SFB-DYRK1A vector containing the N-terminal FLAG-tagged human DYRK1A complementary DNA (cDNA) sequence (NM_001396.4).

Techniques:

Journal: Genetics in medicine : official journal of the American College of Medical Genetics

Article Title: Integrative approach to interpret DYRK1A variants, leading to a frequent neurodevelopmental disorder.

doi: 10.1038/s41436-021-01263-1

Figure Lengend Snippet: Fig. 2 Expression, localization and Tyr321 phosphorylation of DYRK1A mutant proteins. (a) Level of variant DYRK1A proteins expressed in HeLa, HEK293, and COS cells transiently transfected with DYRK1A constructs. Protein levels were normalized on the level of GFP proteins (expressed from a cotransfected pEGFP plasmid). Quantifications were performed on a total of n ≥9 series of cells (n ≥3 Hela cells, n ≥3 HEK293, and n ≥3 COS cells) using ImageJ software. One-way analysis of variance (ANOVA) with multiple comparison test was performed to compare the level of variant DYRK1A proteins to the level of wild‐type DYRK1A protein (orange dashes), applying Bonferroni’s correction. ns not significant; *p < 0.05; **p < 0.01; ***p < 0.001; error bars represent SEM. Green, the variant from gnomAD; red, a truncating variant; gold, the variants tested in this study. (b) DYRK1A’s ability to autophosphorylate on Tyr321 was tested in HEK293 cells (n = 3) by immunoprecipitations with anti-DYRK1A followed by an immunoblot using anti-phospho-HIPK2 as described in Widowati et al. [33]. DYRK1A phospho-Tyr321 levels were normalized with DYRK1A total level (orange dashes). Variant DYRK1A phospho-Tyr321 levels were normalized with total DYRK1A protein levels and expressed as percentage of wild-type level. One-way ANOVA was performed to compare variants to wild-type DYRK1A levels. ns not significant; ***p < 0.001; error bars represent SEM. (c) Immunofluorescence experiment showing that Ser660fs (alias Ser660Profs*43) variant leads to DYRK1A protein aggregation when overexpressed in HeLa cells, using a FLAG-tagged DYRK1A proteins carrying Ser660Profs*43. No aggregation was observed for the Ser660* variant.

Article Snippet: In vitro analysis of variant effect on DYRK1A protein DYRK1A expression plasmids (addgene #101770, Huen lab) were generated from the pMH-SFB-DYRK1A vector containing the N-terminal FLAG-tagged human DYRK1A complementary DNA (cDNA) sequence (NM_001396.4).

Techniques: Expressing, Phospho-proteomics, Mutagenesis, Variant Assay, Transfection, Construct, Plasmid Preparation, Software, Comparison, Western Blot

Journal: Genetics in medicine : official journal of the American College of Medical Genetics

Article Title: Integrative approach to interpret DYRK1A variants, leading to a frequent neurodevelopmental disorder.

doi: 10.1038/s41436-021-01263-1

Figure Lengend Snippet: Fig. 3 DNA methylation signature of DYRK1A loss-of-function (LoF) functionally classifies DYRK1A variants of uncertain significance (VUS). (a) Heatmap showing the hierarchical clustering of discovery DYRK1A LoF cases (n = 10) and age- and sex-matched neurotypical discovery controls (n = 24) used to identify the 402 differentially methylated signature sites shown. Each row corresponds to a CpG site differentially methylated (DM) and the color gradient represents the normalized DNA methylation (DNAm) value from -2.0 (blue) to 2.0 (yellow) at each site. DNA methylation at these sites clearly separate discovery cases (gray) from discovery controls (blue). Euclidean distance metric is used for the clustering dendrogram. (b) Principal components analysis (PCA) visualizing the DNAm profiles of the study cohort at the 402 signature sites. Validation of DYRK1A LoF cases (not used to define the signature sites; red) cluster with discovery cases, while missense (yellow) and distal LoF (green) variants cluster with either cases or controls. individual 33 (Gly486Asp) has an opposite DNAm profile to DYRK1A LoF cases at these sites, suggesting a GoF. (c) Support vector machine (SVM) classification model based on the DNA methylation values in the discovery groups. Each sample is plotted based on its scoring by the model. All samples are clearly positive (>0.5) or negative (<0.5). All DYRK1A validation cases from our cohort (n = 6) classified positively, all control validation cases (n = 94) classified negatively. Missense variants classified clearly positively or negatively (yellow), the distal frameshift variant (individual 18, c.1978del) (green), analyzed in duplicate, classified positively. Pathogenic ARID1B (Coffin–Siris syndrome) and KMT2A (Wiedemann–Steiner syndrome) also classified negatively.

Article Snippet: In vitro analysis of variant effect on DYRK1A protein DYRK1A expression plasmids (addgene #101770, Huen lab) were generated from the pMH-SFB-DYRK1A vector containing the N-terminal FLAG-tagged human DYRK1A complementary DNA (cDNA) sequence (NM_001396.4).

Techniques: DNA Methylation Assay, Methylation, Biomarker Discovery, Plasmid Preparation, Control, Variant Assay

Journal: Genetics in medicine : official journal of the American College of Medical Genetics

Article Title: Integrative approach to interpret DYRK1A variants, leading to a frequent neurodevelopmental disorder.

doi: 10.1038/s41436-021-01263-1

Figure Lengend Snippet: Fig. 4 Summary of the analysis performed to reclassify variants in DYRK1A. Representation of the DYRK1A protein (the kinase domain is indicated in red and the catalytic domain in dark red) with the positions of the different variants tested with the sample ID of the individuals indicated inside the circles. Number: number of individuals with ID reported with the variant; gAD: variant reported in individuals from gnomAD; CSDYRK1A poorly (white), intermediate (gray) or highly (black) evocative, or unknown (-); white star indicates that the individual presents a high CSDYRK1A score but also additional clinical manifestations unusual for DYRK1A syndrome; CADD below 25 (white), between 25 and 30 (gray) or above 30 (black); conservation: highly conserved V = 100%, M > 90%, O > 80% (black), moderately V = 100%, M > 90%, O < 80% (gray) or mildly V = 100% M < 90%, O < 80% (white); Expression or autophosphorylation being normal (white), intermediate decreased (gray), strongly decreased (black); localization was normal (white), affected (gray), or not tested (-); DNA methylation positive (black), negative (white), suggestive of a gain-of-function (GoF) effect (hashed) or not tested (-). Final classification: pathogenic (P), benign/likely benign (B), unknown significance (U).

Article Snippet: In vitro analysis of variant effect on DYRK1A protein DYRK1A expression plasmids (addgene #101770, Huen lab) were generated from the pMH-SFB-DYRK1A vector containing the N-terminal FLAG-tagged human DYRK1A complementary DNA (cDNA) sequence (NM_001396.4).

Techniques: Variant Assay, Expressing, DNA Methylation Assay

Journal: Experimental cell research

Article Title: DYRK1A is required for maintenance of cancer stemness, contributing to tumorigenic potential in oral/oropharyngeal squamous cell carcinoma.

doi: 10.1016/j.yexcr.2021.112656

Figure Lengend Snippet: Fig. 1. Loss of DYRK1A suppresses self-renewal and tumorigenic potential of OSCC. (A) Deletion of DYRK1A was performed in two OSCC cell lines (SCC4 and FaDu) by CRISPR/Cas9 genome editing technique. Deletion of DYRK1A was confirmed by Western blot. GAPDH was used as a loading control. WT; Wild-type, KO; DYRK1A knockout. Arrowhead indicates the DYRK1A-specific band, and asterisk indicates a nonspecific cross-reacting band. (B) Effect of DYRK1A deletion on self-renewal capacity was determined by tumorsphere formation assay. Representative images of tumorspheres formed by the WT and DYRK1A KO cells were shown on the right of each bar graph. Bar indicates 200 μm. Data are means ± SD of triplicate experiment. *P < 0.01 compared to WT. (C) Effect of DYRK1A deletion on anchorage- independent growth ability was determined by soft agar assay. *P < 0.01 compared to WT. (D) Effect of DYRK1A deletion on in vivo tumorigenicity was determined by xenograft tumor assay. FaDu WT and FaDu DYRK1A KO were injected subcutaneously into 5 nude mice. Tumor volumes were measured for 3 weeks. (E) Tumor images and tumor incidence of FaDu WT and FaDu DYRK1A KO were obtained 3 weeks after the injections.

Article Snippet: Exogenous DYRK1A was overexpressed with vector expressing DYRK1A (The pMH-SFB-DYRK1A; Addgene).

Techniques: CRISPR, Western Blot, Control, Knock-Out, Tube Formation Assay, Soft Agar Assay, In Vivo, Injection

Journal: Experimental cell research

Article Title: DYRK1A is required for maintenance of cancer stemness, contributing to tumorigenic potential in oral/oropharyngeal squamous cell carcinoma.

doi: 10.1016/j.yexcr.2021.112656

Figure Lengend Snippet: Fig. 2. Loss of DYRK1A diminishes CSC-enriched ALDH1HIGH population and CSC properties in OSCC. (A) Effect of DYRK1A deletion on ALDH1HIGH cell population in SCC4 and FaDu was determined by Aldefluor assay. Cells were labeled with Aldefluor with and without the ALDH inhibitor DEAB and analyzed by flow cytometry. The gate for ALDH1HIGH cells is determined in relation to the DEAB control (+DEAB) and shows the brightly fluorescent ALDH population versus the side scatter, a population that is absent/decreased in the presence of DEAB. The number shown in each panel reflects the percentage of ALDH1HIGH cells in each cell type. A similar result was obtained from an independent experiment (Supplemental Fig. 2). (B) Effect of DYRK1A deletion on migration ability was determined by transwell migration assay. Migration ability was described as number of migrated cells per field with data as mean ± SD for three randomly selected fields. Representative images of migrated cells from each group were shown on the right of each bar graph. Bar indicates 200 μm *P < 0.01 (C) Effect of DYRK1A deletion on chemo resistance was determined by MTT assay. SCC4 WT and SCC4 DYRK1A KO cells were treated with the indicated concentrations of cisplatin for 2 days, and their viability was determined by MTT assay. **P < 0.05.

Article Snippet: Exogenous DYRK1A was overexpressed with vector expressing DYRK1A (The pMH-SFB-DYRK1A; Addgene).

Techniques: Labeling, Flow Cytometry, Control, Migration, Transwell Migration Assay, MTT Assay

Journal: Experimental cell research

Article Title: DYRK1A is required for maintenance of cancer stemness, contributing to tumorigenic potential in oral/oropharyngeal squamous cell carcinoma.

doi: 10.1016/j.yexcr.2021.112656

Figure Lengend Snippet: Fig. 3. Ectopic DYRK1A expression promotes CSC properties in OSCC. DYRK1A was ectopically overexpressed in SCC9 by transfecting with vector expressing DYRK1A and empty vector (EV) as a control. (A) Ectopic expression of DYRK1A in SCC9 was validated by qPCR (left) and Western blot (right). (B) Effect of DYRK1A overexpression on self-renewal capacity was determined by tumorsphere formation assay. Representative images of tumorspheres formed from each group were shown on the right of the bar graph. *P < 0.01 compared to EV. (C) Effect of DYRK1A overexpression on migration ability was measured by a transwell migration assay. Representative images of migrated cells from each group were shown on the right of the bar graph. *P < 0.05 (D) Effect of DYRK1A overexpression on chemoresistance was determined by MTT assay. The cells were treated with indicated concentrations of cisplatin for 2 days, and their viability was determined. ns, not significant.

Article Snippet: Exogenous DYRK1A was overexpressed with vector expressing DYRK1A (The pMH-SFB-DYRK1A; Addgene).

Techniques: Expressing, Plasmid Preparation, Control, Western Blot, Over Expression, Tube Formation Assay, Migration, Transwell Migration Assay, MTT Assay

Journal: Experimental cell research

Article Title: DYRK1A is required for maintenance of cancer stemness, contributing to tumorigenic potential in oral/oropharyngeal squamous cell carcinoma.

doi: 10.1016/j.yexcr.2021.112656

Figure Lengend Snippet: Fig. 4. DYRK1A is overexpressed in OSCC. (A) Level of DYRK1A mRNA was determined in normal human oral keratinocyte (NHOK) and 9 OSCC cell lines (BapT, FaDu, SCC4, SCC9, SCC15, SCC105, SNU1066, UM6 and YD38) by qPCR. The Ct values of DYRK1A were normalized by the Ct value of GAPDH. (B) Level of DYRK1A protein was determined in NHOK and OSCC cell lines by Western blot analysis. (C) Self-renewal capacity in three groups (high, medium, and low based on their level of DYRK1A protein) was determined by tumorsphere formation assay. (D) ALDH1HIGH cell population in three groups was determined by Aldefluor assay. (E) In vivo DYRK1A expression was determined in normal human oral epithelia (NHOE) and OSCC tissues by immunohistochemical (IHC) staining. (F) Representative examples of DYRK1A IHC staining in NHOE and OSCC tissues in vivo. Bar indicates 100 μm.

Article Snippet: Exogenous DYRK1A was overexpressed with vector expressing DYRK1A (The pMH-SFB-DYRK1A; Addgene).

Techniques: Western Blot, Tube Formation Assay, In Vivo, Expressing, Immunohistochemical staining, Immunohistochemistry

Journal: Experimental cell research

Article Title: DYRK1A is required for maintenance of cancer stemness, contributing to tumorigenic potential in oral/oropharyngeal squamous cell carcinoma.

doi: 10.1016/j.yexcr.2021.112656

Figure Lengend Snippet: Fig. 5. DYRK1A regulates CSC property via FGF2 expression. (A) Effect of DYRK1A deletion on FGF2 expression was assessed in SCC4 and FaDu by qPCR (upper) and Western blot analysis (lower). (B) Effect of DYRK1A deletion on FGF2 secretion was determined by ELISA. (C) Effect of DYRK1A overexpression on FGF2 expression was assessed in SCC9 by qPCR (upper) and Western blot analysis (lower). (D) Effect of DYRK1A overexpression on FGF2 secretion was determined by determined by ELISA. (E) Effect of FGF2 on self-renewal capacity of SCC4 WT and DYRK1A KO was measured by tumorsphere formation assay. The assays were performed in the absence and presence of FGF2 (50, 100 and 200 ng/ml). *P < 0.05 compared to untreated control cells. (F) Effect of FGF2 on migration ability of SCC4 WT and DYRK1A KO was measured by transwell migration assay. The assays were performed in the absence and presence of FGF2. (G) Effect of FGF2 neutralizing antibody on self-renewal capacity of DYRK1A-overerxpressing SCC9 was determined by tumorsphere assay. The assays were performed in the absence and presence of FGF2 antibody (1, 2.5, and 5 ng/ml). *P < 0.05 and **P < 0.01 compared to untreated control cells. (H) Effect of FGF2 neutralizing antibody on migration ability of DYRK1A-overerxpressing SCC9 was measured by transwell migration assay. The assays were performed in the absence and presence of FGF2 antibody.

Article Snippet: Exogenous DYRK1A was overexpressed with vector expressing DYRK1A (The pMH-SFB-DYRK1A; Addgene).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Over Expression, Tube Formation Assay, Control, Migration, Transwell Migration Assay

Journal: Experimental cell research

Article Title: DYRK1A is required for maintenance of cancer stemness, contributing to tumorigenic potential in oral/oropharyngeal squamous cell carcinoma.

doi: 10.1016/j.yexcr.2021.112656

Figure Lengend Snippet: Fig. 6. Expression of DYRK1A and FGF2 are elevated in CSC-enriched populations, and their levels are positively correlated in OSCC cells. (A) Cisplatin-resistant SCC4 (CR-SCC4) cells were isolated from SCC4 treated with 25 μM cisplatin for 2 days. The IC50 value for cisplatin in SCC4 and CR-SCC4 was determined by MTT assay. Cells were treated with various concentrations of cisplatin for 48 h, and their viability was determined. (B) Self-renewal capacity of SCC4 and CR-SCC4 was determined by tumorsphere formation assay. *P < 0.05 and **P < 0.01 compared to SCC4. (C) Expression of DYRK1A and FGF2 was assessed in SCC4 and CR- SCC4 by qPCR (left) and Western blot (right). (D) ALDH1HIGH (CSC-enriched population) and ALDH1low (non-CSC population) cell populations were sorted from SCC4 cells by flow cytometry. Expression of DYRK1A and FGF2 was assessed in ALDH1low and ALDH1HIGH cell populations by qPCR. (E) Expression of DYRK1A and FGF2 was assessed in tumorspheres (S) and their corresponding adherent monolayer cells (M) derived from multiple OSCC cell lines by qPCR. (F) Correlation analysis of DYRK1A and FGF2 mRNA was determined based on their expression levels in 14 human SCC cell lines by qPCR.

Article Snippet: Exogenous DYRK1A was overexpressed with vector expressing DYRK1A (The pMH-SFB-DYRK1A; Addgene).

Techniques: Expressing, Isolation, MTT Assay, Tube Formation Assay, Western Blot, Flow Cytometry, Derivative Assay

Journal: medRxiv

Article Title: Integrative approach to interpret DYRK1A variants, leading to a frequent neurodevelopmental disorder

doi: 10.1101/2021.01.20.21250155

Figure Lengend Snippet:

Article Snippet: DYRK1A expression plasmids were generated from the pMH-SFB- DYRK1A vector containing the human DYRK1A cDNA sequence (NM_001396.4) tagged with FLAG peptide at the N-Terminal side (purchased from addgene #101770; Huen lab).

Techniques: Translocation Assay, Sequencing, Hybridization, DNA Methylation Assay

Journal: medRxiv

Article Title: Integrative approach to interpret DYRK1A variants, leading to a frequent neurodevelopmental disorder

doi: 10.1101/2021.01.20.21250155

Figure Lengend Snippet: (A) Clinical score out of 20 points established according to the most recurrent clinical features presented by patients (the weight assigned to each symptom being based on its recurrence): clinical symptoms are out of 15 points, while the facial appearance is out of 5 points. EV: enlarged ventricules; CCA/H: corpus callosum agenesis or hypoplasia, CA: cerebral atrophy, CeA: cerebellar atrophy ( B) Clinical scores calculated for individuals carrying pathogenic variants in DYRK1A reported here and for whom photographs were available (n=21)(initial cohort, DYRK1A_I, scores 13-17.5 with a mean of 15.5), the previously published individuals (replication cohort, DYRK1A_R, scores 13.5-18, mean=15.3) and the individuals affected with other frequent monogenic forms of ID, associated to mutations in ANKRD11, MED13L, DDX3X, ARID1B, SHANK3, TCF4 or KMT2A (scores 3-12.5, mean=7). The clinical score for the individuals carrying missense or distal frameshift variants are indicated in yellow (test); The threshold of CS DYRK1A >=13 appeared to be discriminant between individuals with LoF variants in DYRK1A (all ≥13) and individuals suffering from another form of ID (all<13) and a score above this threshold was therefore considered “highly suggestive”. We classified individuals with CS DYRK1A <10 as “poorly evokative” and individuals with a CS DYRK1A comprised between 10 and 13 as “intermediate”. Brown-Forsythe and Welsh ANOVA tests with Dunnett’s T 3 multiple comparisons test were performed. ns: not significant; ** p<0.01; ***< p<0.001, error bars represent SD.

Article Snippet: DYRK1A expression plasmids were generated from the pMH-SFB- DYRK1A vector containing the human DYRK1A cDNA sequence (NM_001396.4) tagged with FLAG peptide at the N-Terminal side (purchased from addgene #101770; Huen lab).

Techniques:

Journal: medRxiv

Article Title: Integrative approach to interpret DYRK1A variants, leading to a frequent neurodevelopmental disorder

doi: 10.1101/2021.01.20.21250155

Figure Lengend Snippet: (A) Level of variant DYRK1A proteins expressed in HeLa, HEK293 and COS cells transiently transfected with DYRK1A constructs. Protein levels were normalized on the level of GFP proteins (expressed from a cotransfected pEGFP plasmid). Quantifications were performed on a total of n >=9 series of cells (n>=3 Hela cells, n>= 3 HEK293 and n >= 3 COS cells). One-way ANOVA with multiple comparison test was performed to compare the level of variant DYRK1A proteins to the level of wild[type DYRK1A protein, applying Bonferroni’s correction: ns: not significant; *p < 0.05; **p < 0.01;***p<0.001; error bars represent SEM, standard error of the mean, ( B) DYRK1A’s ability to autophosphorylate on Tyr321 was tested in HEK293 cells (n=3) by immunoprecipitations with anti-DYRK1A followed by an immunoblot using an anti-HIPK2 as described in Widowati et al. DYRK1A phospho-Tyr321 levels were normalized with DYRK1A total level. Variant DYRK1A phospho-Tyr321 levels were normalized with total DYRK1A protein levels and expressed as percentage of wild-type level. One-way ANOVA test was performed to compare variants to wild-type DYRK1A levels. ns: not significant; ***p<0.001; error bars represent SEM, standard error of the mean (C) Immunofluorescence experiment showing that Ser660fs (alias Ser660Profs*43) variant leads to DYRK1A protein aggregation when overexpressed in HeLa cells, using a FLAG-tagged DYRK1A proteins carrying Ser660Profs43. No aggregation was observed for the Ser660* variant.

Article Snippet: DYRK1A expression plasmids were generated from the pMH-SFB- DYRK1A vector containing the human DYRK1A cDNA sequence (NM_001396.4) tagged with FLAG peptide at the N-Terminal side (purchased from addgene #101770; Huen lab).

Techniques: Variant Assay, Transfection, Construct, Plasmid Preparation, Western Blot, Immunofluorescence

Journal: medRxiv

Article Title: Integrative approach to interpret DYRK1A variants, leading to a frequent neurodevelopmental disorder

doi: 10.1101/2021.01.20.21250155

Figure Lengend Snippet: (A) Heatmap showing the hierarchical clustering of discovery DYRK1A LoF cases (n[=[10) and age- and sex-matched neurotypical discovery controls (n[=[24) used to identify the 402 differentially methylated signature sites shown. The color gradient represents the normalized DNA methylation value from -2.0 (blue) to 2.0 (yellow) at each site. DNA methylation at these sties clearly separate discovery cases (grey) from discovery controls (blue). Euclidian distance metric is used for the clustering dendrogram. (B) Principal components analysis (PCA) visualizing the DNAm profiles of the study cohort at the 402 signature sites. Validation of DYRK1A LoF cases (not used to define the signature sites; red) cluster with discovery cases, while missense (yellow) and distal LoF (green) variants cluster with either cases or controls. Ind #33 (Gly486Asp) has an opposite DNAm profile to DYRK1A LoF cases at these sites, suggesting a GoF. (C) Support vector machine (SVM) classification model based on the DNA methylation values in the discovery groups. Each sample is plotted based on its scoring by the model. All samples are clearly positive (>0.5) or negative (<0.5). All DYRK1A validation cases from our cohort (n=6) classified positively, all control validation cases (n=94) classified negatively. Missense variants classified clearly positively or negatively, the distal frameshift variant (Ind #18, c.1978del), analyzed in duplicate, classified positively. Pathogenic ARID1B (Coffin-Siris syndrome) and KMT2A (Wiedemann Steiner syndrome) also classified negatively.

Article Snippet: DYRK1A expression plasmids were generated from the pMH-SFB- DYRK1A vector containing the human DYRK1A cDNA sequence (NM_001396.4) tagged with FLAG peptide at the N-Terminal side (purchased from addgene #101770; Huen lab).

Techniques: Methylation, DNA Methylation Assay, Plasmid Preparation, Variant Assay

Journal: medRxiv

Article Title: Integrative approach to interpret DYRK1A variants, leading to a frequent neurodevelopmental disorder

doi: 10.1101/2021.01.20.21250155

Figure Lengend Snippet: Representation of the DYRK1A protein (the kinase domain is indicated in red and the catalytic domain in dark red) with the positions of the different variants tested with the sample #ID of the individuals indicated inside the circles. Number: number of individuals with ID reported with the variant; gAD: variant reported in individuals from gnomAD; CS DYRK1A poorly (white), intermediate (grey) or highly (black) evocative, or unknown (-); CADD below 25 (white), between 25 and 30 (grey) or above 30 (black); conservation: highly conserved V=100%, M>90%, O>80% (black), moderately V=100%, M>90%, O<80% (grey) or midly V=100% M<90%, O<80% (white); Expression or autophosphorylation being normal (white), intermediate decreased (grey), strongly decreased (black); Localization was normal (white), affected (grey) or not tested (-); DNAmethylation positive (black), negative (white), suggestive of a GoF effect (hashed) or not tested (-). Final classification: Pathogenic (P), Benign (B), Unknown significance (U).

Article Snippet: DYRK1A expression plasmids were generated from the pMH-SFB- DYRK1A vector containing the human DYRK1A cDNA sequence (NM_001396.4) tagged with FLAG peptide at the N-Terminal side (purchased from addgene #101770; Huen lab).

Techniques: Variant Assay, Expressing