Journal: medRxiv

Article Title: Ultra-rare biallelic THAP12 variants cause loss of function and underlie severe epileptic encephalopathy

doi: 10.64898/2026.02.27.26347078

Figure Lengend Snippet: (A) Sanger sequencing confirms compound heterozygous variants in THAP12 in both affected siblings, with a maternally inherited frameshift (c.312del, red arrow) and a paternally inherited missense variant (c.829C>A, black arrow). (B) The two variants affect conserved residues within protein domains, particularly a proline at position 277 in the DUF4371 domain, as shown in a multi-species alignment (red arrow; Hs: Homo sapiens; Mm: Mus musculus; Rn: Rattus norvegicus; Xt: Xenopus tropicalis; Dr: Danio rerio ). (C) A Sashimi plot of RNA-seq reads from patients’ primary fibroblasts across the THAP12 locus shows no major changes in exon usage or alternative splicing between probands and their parents. Read counts on the splice junction arcs indicate the number of split reads supporting each exon-exon connection in each sample. (D) Structural modelling with AlphaFold3 predicts that THAP12 forms homodimers primarily through interactions between DUF4371 domains (left). In a THAP12-DNA complex predicted using AlphaFold3, the N-terminal THAP zinc-finger domains interacts with DNA using an electropostive surface (right). (E) The maternally inherited frameshift variant (Glu105AsnfsTer2) is predicted to truncate the protein after residue 106, abolishing the DUF4371 domain and likely impairing dimerization. The N-terminal segment (residues 1-106) is shown in color, corresponding to the truncated product of the frameshift allele. The paternally inherited Pro277Thr missense variant affects a conserved residue buried within the hydrophobic core of the DUF4371 domain, likely disrupting local folding and protein stability. (F-G) THAP12 transcript levels are not significantly changed in patient fibroblasts compared to parental controls, as shown by RNA-seq and qPCR analyses. (H-I) In contrast, THAP12 protein levels in patient fibroblasts are significantly reduced in both probands, as shown by Western blot and quantification. (J) Volcano plot displaying differentially expressed genes from bulk RNA-sequencing analysis of patient-derived fibroblasts compared to parental controls. Downregulated genes include several involved in neuronal and synaptic function (e.g., TUBB, RIMS1, GABRA3, KCNMB1 ). Significance is color-coded according to the - log10(adjusted p-value). The full list of differentially expressed genes is provided in Table S1. (K) Pathway enrichment analysis of differentially expressed genes highlights over-representation of pathways such as “Neuronal System”, “Signal Transduction”, and “Transmission across Chemical Synapses”. Dot size indicates the number of genes in each pathway; color represents -log10(p-value). Statistical analyses in panels F, G, and I used unpaired two-tailed Student’s t-test: *p < 0 . 05, **p < 0 . 01, ***p < 0 . 001, ****p < 0 . 0001; ns, not significant .

Article Snippet: Whole genome sequencing and analysis and RNA sequencing data were provided the Broad Institute Center for Mendelian Genomics (CMG) and were funded by the National Human Genome Research Institute (NHGRI) grants UM1HG008900 (with additional support from the National Eye Institute, and the National Heart, Lung and Blood Institute), R01HG009141, U01HG011755, and in part by the Chan Zuckerberg Initiative Donor-Advised Fund at the Silicon Valley Community Foundation grants 2019-199278, 2020-224274 (https://doi.org/10.37921/236582yuakxy) (funder DOI 10.13039/100014989).

Techniques: Sequencing, Variant Assay, RNA Sequencing, Alternative Splicing, Residue, Western Blot, Derivative Assay, Transduction, Transmission Assay, Two Tailed Test

Journal: medRxiv

Article Title: Ultra-rare biallelic THAP12 variants cause loss of function and underlie severe epileptic encephalopathy

doi: 10.64898/2026.02.27.26347078

Figure Lengend Snippet: (A) Schematic comparison of the human variants identified in the two affected siblings (top) with the corresponding genetically engineered mouse alleles (bottom), showing equivalent positions across conserved domains. (B) Structural alignment of the mouse and human THAP12 proteins, whose structures were predicted using AlphaFold3, reveals high overall similarity and highlights the position of the conserved Pro273 residue (orthologous to human Pro277) within a similar hydrophobic core of the DUF4371 domain. (C) Sanger sequencing confirms the genotypes of the three engineered alleles in mice (red arrows): insT, Glu105Asnfs*2 and Pro273Thr . (D) Representative images of E12.5 mouse embryos show that homozygous Pro273Thr mutants are severely developmentally delayed compared to wild-type and heterozygous littermates. (E) Table summarizing genotype frequencies at different developmental stages in intercrosses between heterozygous carriers. The absence of homozygous animals at birth confirms early embryonic lethality. Of note, data from both insT and Glu105Asnfs*2 alleles are gathered as “KO” in this panel. Full data for each stage and genotype are provided in Table S2. (F) Western blot of E12.5 embryonic heads shows reduced THAP12 protein levels in Pro273Thr homozygous mutants. (G) Quantification of THAP12 signal relative to wild-type confirms a significant decrease in homozygous embryos. Data are expressed as fold change relative to wild-type levels. Statistical analyses in panel G used a o ne-way ANOVA with Tukey post-hoc test: *p < 0 . 05, **p < 0 . 01, ***p < 0 . 001 .

Article Snippet: Whole genome sequencing and analysis and RNA sequencing data were provided the Broad Institute Center for Mendelian Genomics (CMG) and were funded by the National Human Genome Research Institute (NHGRI) grants UM1HG008900 (with additional support from the National Eye Institute, and the National Heart, Lung and Blood Institute), R01HG009141, U01HG011755, and in part by the Chan Zuckerberg Initiative Donor-Advised Fund at the Silicon Valley Community Foundation grants 2019-199278, 2020-224274 (https://doi.org/10.37921/236582yuakxy) (funder DOI 10.13039/100014989).

Techniques: Comparison, Residue, Sequencing, Western Blot

Journal: medRxiv

Article Title: Ultra-rare biallelic THAP12 variants cause loss of function and underlie severe epileptic encephalopathy

doi: 10.64898/2026.02.27.26347078

Figure Lengend Snippet: (A-B) Dorsal views of Tg[ elavl3 :GFP] larvae at 2 dpf (A) and 5 dpf (B) showing a reduced brain size in thap12 CRISPant compared to sham-injected controls. The lack of a clear midbrain-hindbrain boundary (dotted line) is indicated by asterisks. (C-E) Transverse sections immunostained for elavl3 show reduced brain size in thap12 CRISPant embryos at 2 dpf ( C ) and at 5 dpf ( D, E ). (F) Dorsal view of 3 dpf brains immunostained for acetylated tubulin showing reduced density of axonal tracts in thap12 CRISPant , especially at the level of the commissure (asterisks). (G-H) Quantification of brain area from whole-brain imaging of Tg[elavl3:GFP] larvae at 2 dpf (G) and 5 dpf (H) confirms a significant reduction of brain size in thap12 CRISPant . (I-J) thap12 CRISPant larvae show reduced numbers of elavl3 + neurons from immunolablled cross-sections (I) and commissural axonal tracts (J). (K) Volcano plot displaying differentially expressed genes from bulk RNA-sequencing analysis of microdissected larval brains from 4dpf thap12a -/- compared to wild-type siblings. Upregulated genes include several involved in apoptosis (e.g. tp53 ) and cell cyle (e.g. ccng1 ). Significance is color-coded according to the -log10(adjusted p-value). (L) Pathway enrichment analysis identifies p53 signaling, cell cycle, and metabolic stress as significantly enriched pathways in mutants compared to wild-type siblings. Dot size indicates the number of genes in each pathway; color represents -log10(p-value). (M) Acridine orange staining reveals increased cell death in the brain of thap12 CRISPant at 1 dpf. (N) Anti-phospho-H3 immunostaining shows a reduced number of proliferating cells in thap12 CRISPRant larvae at 1 dpf. Quantification are shown in panel O and P. (Q-T) Injection of human wild-type THAP12 mRNA, but not Pro277Thr mutant mRNA, rescues reduced proliferation in thap12 CRISPant larvae at 1 dpf (Q-R) and partially rescues brain size at 2 dpf (S-T) . The lack of a clear midbrain-hindbrain boundary (dotted line) described in panel A is indicated by asterisks. Scale bars are shown on each panel. Statistical analyses in panel G-J and O-P used unpaired two-tailed Student’s t-test, and in panels R and T used one-way ANOVA: *p < 0 . 05, **p < 0 . 01, ***p < 0 . 001, ****p < 0 . 0001; ns, not significant. fb: forebrain; mb: midbrain; hb: hindbrain; mhb: midbrain-hindbrain boundary (dotted line); bs: brainstem; ey: eye; re: retina; tec: tectum; teg: tegmentum; hl: hypothalamus .

Article Snippet: Whole genome sequencing and analysis and RNA sequencing data were provided the Broad Institute Center for Mendelian Genomics (CMG) and were funded by the National Human Genome Research Institute (NHGRI) grants UM1HG008900 (with additional support from the National Eye Institute, and the National Heart, Lung and Blood Institute), R01HG009141, U01HG011755, and in part by the Chan Zuckerberg Initiative Donor-Advised Fund at the Silicon Valley Community Foundation grants 2019-199278, 2020-224274 (https://doi.org/10.37921/236582yuakxy) (funder DOI 10.13039/100014989).

Techniques: Injection, Imaging, RNA Sequencing, Staining, Immunostaining, Mutagenesis, Two Tailed Test

Journal: Frontiers in Cell and Developmental Biology

Article Title: Establishment of human periodontal ligament cell lines with ALPL mutations to mimic dental aspects of hypophosphatasia

doi: 10.3389/fcell.2025.1572571

Figure Lengend Snippet: Potential consequences of genetic changes in clone 1.5. and investigation of mRNA products via RT-PCR. (A) Sequencing of clone 1.5 gDNA showed a huge genetic alteration within the ALPL exon 6 locus, including a 48 bp duplication and a 48 bp deletion. Clonal cDNA sequencing further clarified that the deletion results in an in-frame loss of 48 bp, thereby subsequently should result in loss of 16 aa within the corresponding TNAP protein domain. The corresponding insertion is not detected in the cDNA sequencing. (B) A potential gain of a novel splice acceptor site was predicted by different algorithms (Alamut Visual Plus splice summary is shown, blue box marks ALPL exon 6 locus, red box indicates gained sequence, green box marks novel splice site). (C) The in silico predicted novel splice product was detectable via RT-PCR by electrophoresis. The analyses indicate an additional PCR product in clone 1.5. While the larger product (marked with white arrowhead) is also present in clone 1.3, the smaller band was only detected in clone 1.5 (marked with green arrowhead). (D) Sequencing of the smaller RT-PCR products shows deletion of 48 bp and the active usage of the new splice site. (E) Schematic presentation of sequence aberration and splice prediction for the wildtype and the aberrant sequence on gDNA (upper panel) and mRNA level (lower panel). PCR primer binding sites and expected RT-PCR product sites (in grey) are in addition sketched below. The CRISPR target region is marked in red. The green box marks 48 bp duplication in clone 1.5, while the crossed white box indicates deletion of 48 bp. Black asterisk marks position of potential novel splice site.

Article Snippet: Prediction of splice site consequences after the 48 bp duplication via Alamut Visual Plus software (combining four different splice algorithms) resulted in identification of a high-scoring novel splice acceptor site in this region ( ; SpliceSite Finder score: 81.9; MaxEntScan score: 7.2; NNSPLICE score: 0.9; GeneSplicer score: 8.4).

Techniques: Reverse Transcription Polymerase Chain Reaction, Sequencing, In Silico, Electrophoresis, Binding Assay, CRISPR

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Accounting for ALA Natural Mutations Enhances the Efficiency of Graphene Oxide Nanopriming in Bar-Modified Arabidopsis.

doi: 10.1002/advs.202500058

Figure Lengend Snippet: Figure 5. Genome-wide DNA methylation analysis in WT and GM seeds before and after GO treatments. a) Circos plots show the methylation levels across different chromosomes for WT and GM seeds before and after GO exposure. The colored sections on the outer ring represent different chromo- somes. The concentric rings from outer to inner illustrate untreated samples, 0.75 mg-C/L GO-treated samples, and 1.5 mg-C/L GO-treated samples. Whole-genome methylation levels were calculated as mean values within 100 kb windows for each chromosome. The color gradient from cyan to red indicates the methylation percentages, whereas the innermost ring represents gene density. b) Whole-genome methylation levels in CG, CHG, and CHH contexts for WT and GM seeds after various GO exposures (sample size n = 3). Data are presented as means ± SD, with gray and red dots depicting individual data points in the WT and GM groups. Three biological replicates were included for each treatment. Independent sample two-sided t-tests were performed: * indicates significant differences within the same genotype relative to the condition without GO, and # indicates significant differences between GM and WT under the same treatment. Significant differences are marked with p values. c) Differential methylation regions at promoter regions and gene bodies of key genes (Figure 1c) are represented by a color gradient showing Log2FC in methylation levels. Triangles and circles indicate CG and CHH methylation, respectively. No differential methylation was observed in the CHG context. Regions without differential methylation are blank. d) Dot plots of differential methylation for ALA metabolism-related genes compare promoter and gene body methylation across conditions. Blue and red dots denote different comparisons, as indicated in the legend. Dots with circles indicate methylation levels with |Log2FC| >10.

Article Snippet: Library construction was performed using an MGIEasy whole-genome bisulfite library preparation kit (MGI).

Techniques: Genome Wide, DNA Methylation Assay, Methylation