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-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