Am J Hum Genet

ATOH1 encodes a basic helix-loop-helix transcription factor critical for hindbrain development and mechanosensory system formation. While animal models have provided extensive functional insights, few human disease-causing variants in ATOH1 have been reported and with no clear functional validation. Here, we report three heterozygous frameshift variants identified in five unrelated families, leading to C-ter truncations of ATOH1 and consistently associated with hearing loss, subtle motor impairments, and a highly recognizable pattern of brainstem malformations. Diffusion tensor imaging in two individuals further revealed reproducible anomalies in specific fiber tracts, supporting a convergent neuroanatomical signature. We also report an early-truncating variant, which, in contrast, is recessive and causes a distinct neurodevelopmental syndrome with highly severe cerebellar and pontine hypoplasia. Functional assays demonstrate that, unlike recessive variants, C-terminal truncating variants retain transcriptional activity but display increased protein stability. In vivo modeling using zebrafish showed that C-terminal truncations of atoh1a are sufficient to disrupt hindbrain neurogenesis and lateral-line hair cell specification. Furthermore, comparisons with loss-of-function phenotypes support a gain-of-function mechanism. Altogether, our findings establish that dominant and recessive ATOH1 variants give rise to different neurodevelopmental syndromes through distinct pathological mechanisms. Our work also underscores the importance of tight temporal control of transcription factor activity during hindbrain development and demonstrates how even subtle neurological phenotypes can arise from early disruption of core developmental programs.