Genetic and protein interaction studies between the ciliary dyslexia candidate genes DYX1C1 and DCDC2

**Background:** Developmental dyslexia (DD) is the most common neurodevelopmental learning disability with established heritability. DYX1C1 (DNAAF4) and DCDC2 are among the most replicated dyslexia candidate genes, both implicated in neuronal migration, cilia function, and cytoskeletal interactions. Mutations in DYX1C1 cause primary ciliary dyskinesia, while DCDC2 loss-of-function mutations cause nephronophthisis-related ciliopathies, hearing loss, and neonatal sclerosing cholangitis. CPAP (CENPJ), a centrosomal protein linked to microcephaly and cilia regulation, was previously shown to interact with DYX1C1. Given these overlapping roles, the authors hypothesized a functional interaction between DYX1C1 and DCDC2.

**Methods:** The study employed multiple experimental approaches: (1) Endogenous co-immunoprecipitation (co-IP) in HeLa cell extracts and 15-day-old hiPSC-derived brain organoids to test protein-protein interactions; (2) Domain-mapping using deletion constructs (DYX1C1ΔTPR, DYX1C1Δp23, DYX1C1ΔDYX) transfected into doxycycline-inducible CPAP-GFP hTERT-RPE1 cells followed by GFP-trap pulldown; (3) Immunofluorescence confocal microscopy in hTERT-RPE1 cells to assess subcellular co-localization; (4) Zebrafish morpholino (MO) co-injection studies using subphenotypic doses (20 µM dyx1c1 MO + 100 µM dcdc2b MO) with standard control MO to maintain uniform concentration, examining ciliary phenotypes at 2 days post-fertilization (dpf); (5) siRNA knockdown of DYX1C1 and DCDC2 in hTERT-RPE1 cells followed by qRT-PCR to assess mutual transcriptional regulation; (6) qRT-PCR on RNA from dyx1c1 morphant zebrafish at 1 dpf and 2 dpf.

**Key Results:** (1) Endogenous co-IP confirmed protein-protein interactions between CPAP and both DCDC2 and DYX1C1 in HeLa cells, with CEP152 as positive control and CEP350 as negative control. The DYX1C1-CPAP interaction was also confirmed in brain organoids by reciprocal immunoprecipitation. (2) Domain mapping revealed that deletion of the p23 domain (DYX1C1Δp23) abrogated the interaction with CPAP, while deletions of TPR or DYX domains did not. (3) Immunofluorescence showed DYX1C1 co-occurrence with CPAP at the centrosome in hTERT-RPE1 cells. DCDC2 co-occurred with CPAP at centrosomes during metaphase but not during cytokinesis. (4) In zebrafish, individual injection of 20 µM dyx1c1 MO or 100 µM dcdc2b MO produced no ciliary phenotype. However, co-injection of both at these subphenotypic doses resulted in a dramatic exacerbation of the ciliary phenotype (strong curved body axis) in 60% of embryos (n=100 per group). (5) siRNA knockdown of DYX1C1 in hTERT-RPE1 cells significantly reduced DCDC2 expression (p<0.05, t-test), and knockdown of DCDC2 significantly reduced DYX1C1 expression (p<0.05, t-test). Knockdown efficiency was confirmed (p<0.0001 for both). (6) In zebrafish, dcdc2b expression was unchanged in dyx1c1 morphants at 1 dpf (p=0.2597) and 2 dpf (p=0.4521).

**Clinical Implications:** This study establishes the first direct evidence of physical and functional interaction between DYX1C1 and DCDC2, two major dyslexia candidate genes, mediated in part through the centrosomal protein CPAP. The synergistic genetic interaction in zebrafish suggests that combined perturbations in these genes may produce more severe phenotypes than individual defects, which could explain variable expressivity in human ciliopathies and dyslexia. The mutual transcriptional regulation observed in human cells (but not whole zebrafish) suggests tissue-specific regulatory mechanisms. These findings contribute to the growing understanding that dyslexia-associated genes function in ciliary and centrosomal pathways, potentially linking neurodevelopmental disorders to ciliary biology. The authors note limitations including the need for rescue experiments in zebrafish, further domain mapping for DCDC2-CPAP interaction, and confirmation of transcriptional regulation via overexpression of knockdown-resistant constructs. Future studies combining genetic, behavioral, and molecular approaches in patient cohorts and model systems are needed to provide an integrative picture of ciliary dyslexia candidate genes in health and disease.