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

Article Title: De novo variants in SP9 cause a novel form of interneuronopathy characterized by intellectual disability, autism spectrum disorder, and epilepsy with variable expressivity.

doi: 10.1016/j.gim.2024.101087

Figure Lengend Snippet: Figure 4 SP9 pathogenic variants are located in DNA binding sites of the protein. A. Characteristic hallmarks of SP/KLF family members. Consensus sequences for the zinc-finger domains of all the SP and KLF transcription factors in human (25 transcription factors), Drosophila (9 transcription factors), and C. elegans (6 transcription factors) are shown for the SP transcription factors, the KLF transcription factors, and the entire family. All DNA-binding domains comprise 81 aa, with the exception of those of Ce-Y40B1A.4 (finger 1: CXXC instead of CXXXXC), Ce-T22C8.5 (finger 3: HXXXXH instead of HXXXH), and D-BTD (finger 2: CXXC instead of CXXXXC). The highly conserved BTD box located at the N terminus of the zinc fingers is a unique feature of the SP transcription factors. Bold capital letters indicate residues that are 100% conserved between all family members (black), between all KLF transcription factors (green), or between all SP transcription factors (purple). Capital letters indicate N90% conservation and lowercase letters N75% conservation. Blue bars indicate cysteine and histidine residues implicated in zinc coordination. Turquoise bars are supposed to be in contact with DNA. Arrows point to residues that probably determine C2H2 recognition by specific contacts with DNA bases. (Adapted from G Suske. Genomics 2005). B. Position of missense (in gray) and frameshift (in black) variants along SP9 sequence. C. Fluorescence microscopy images of T98G cells transfected with the indicated SP9 constructs. D. Effects of SP9 mutants on its binding to candidate regions uc462, uc463, uc467, and uc468 of ARX and subsequent activation of luciferase reporter gene transcription. Significant differences (n = 3) were assessed by one-way analysis of variance with Dunnett’s post hoc statistical test: **P < .01, ***P < .001, ****P < .0001. E. Percentage of amino acids per SP9 domain that feature at least 1 missense gnomAD variant. The P values obtained by χ2 for the comparison of variant proportions between the different regions of SP9 are shown.

Article Snippet: All SP9 mutants were generated using standard procedures from the Myc-DDK-tagged-Human SP9 plasmid (Origene, RC227808) using the CloneAmp HiFi PCR premix (Takara Bio, catalog no. 639298), Cloning Enhancer (Takara Bio, catalog no. 639615), and In-Fusion HD Cloning Plus Kits (Takara Bio, catalog no. 638910) according to the manufacturer’s instructions and using primers listed in Supplemental Table 2.

Techniques: Binding Assay, Sequencing, Fluorescence, Microscopy, Transfection, Construct, Activation Assay, Luciferase, Variant Assay, Comparison

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

Article Title: De novo variants in SP9 cause a novel form of interneuronopathy characterized by intellectual disability, autism spectrum disorder, and epilepsy with variable expressivity.

doi: 10.1016/j.gim.2024.101087

Figure Lengend Snippet: Figure 4 SP9 pathogenic variants are located in DNA binding sites of the protein. A. Characteristic hallmarks of SP/KLF family members. Consensus sequences for the zinc-finger domains of all the SP and KLF transcription factors in human (25 transcription factors), Drosophila (9 transcription factors), and C. elegans (6 transcription factors) are shown for the SP transcription factors, the KLF transcription factors, and the entire family. All DNA-binding domains comprise 81 aa, with the exception of those of Ce-Y40B1A.4 (finger 1: CXXC instead of CXXXXC), Ce-T22C8.5 (finger 3: HXXXXH instead of HXXXH), and D-BTD (finger 2: CXXC instead of CXXXXC). The highly conserved BTD box located at the N terminus of the zinc fingers is a unique feature of the SP transcription factors. Bold capital letters indicate residues that are 100% conserved between all family members (black), between all KLF transcription factors (green), or between all SP transcription factors (purple). Capital letters indicate N90% conservation and lowercase letters N75% conservation. Blue bars indicate cysteine and histidine residues implicated in zinc coordination. Turquoise bars are supposed to be in contact with DNA. Arrows point to residues that probably determine C2H2 recognition by specific contacts with DNA bases. (Adapted from G Suske. Genomics 2005). B. Position of missense (in gray) and frameshift (in black) variants along SP9 sequence. C. Fluorescence microscopy images of T98G cells transfected with the indicated SP9 constructs. D. Effects of SP9 mutants on its binding to candidate regions uc462, uc463, uc467, and uc468 of ARX and subsequent activation of luciferase reporter gene transcription. Significant differences (n = 3) were assessed by one-way analysis of variance with Dunnett’s post hoc statistical test: **P < .01, ***P < .001, ****P < .0001. E. Percentage of amino acids per SP9 domain that feature at least 1 missense gnomAD variant. The P values obtained by χ2 for the comparison of variant proportions between the different regions of SP9 are shown.

Article Snippet: All SP9 mutants were generated using standard procedures from the Myc-DDK-tagged-Human SP9 plasmid (Origene, RC227808) using the CloneAmp HiFi PCR premix (Takara Bio, catalog no. 639298), Cloning Enhancer (Takara Bio, catalog no. 639615), and In-Fusion HD Cloning Plus Kits (Takara Bio, catalog no. 638910) according to the manufacturer’s instructions and using primers listed in Supplemental Table 2.

Techniques: Binding Assay, Sequencing, Fluorescence, Microscopy, Transfection, Construct, Activation Assay, Luciferase, Variant Assay, Comparison