# Human neuronal networks on micro-electrode arrays as a tool to assess genotype-phenotype correlation in CACNA1A-related disorders

## TL;DR

This study uses human neurons to investigate how genetic changes in CACNA1A affect brain activity and help classify uncertain genetic variants.

## Contribution

The study introduces a novel method using patient-derived and CRISPR-engineered human neuronal networks to assess CACNA1A variant effects.

## Key findings

- CACNA1A haploinsufficiency caused subtle changes in glutamatergic network activity.
- Missense variants had a more pronounced effect on overall network function.
- Three variants of uncertain significance were classified as likely pathogenic based on network alterations.

## Abstract

CACNA1A-related disorders constitute a diverse group of neurological conditions, including ataxia, migraine, and epilepsy. Despite extensive genetic studies, clear genotype-phenotype correlations remain elusive. Moreover, next-generation sequencing has identified many variants of uncertain significance (VUS). Here, we leveraged patient-derived and CRISPR-Cas9-engineered human neuronal networks to explore relationships between CACNA1A variants and neurophysiological activity. CACNA1A haploinsufficiency induced subtle alterations in glutamatergic network activity, whereas missense variants had a more pronounced effect on overall network function. Network fingerprints were most affected from patients where ataxia co-occurred with migraine or epilepsy. Furthermore, we analyzed the impact of CRISPR-Cas9-induced VUS on network developmental trajectories. Although functional changes could not be directly linked to clinical phenotypes, all tested variants induced measurable alterations in neuronal network function, supporting their classification as likely pathogenic. These findings highlight the potential of human neuronal networks as a translational model for evaluating CACNA1A variant effects and improving clinical variant interpretation.

•CACNA1A patient-derived human neuronal networks showed variant-specific alterations•A gain-of-function variant linked to epilepsy caused fragmented synchronization•This variant also led to an early increased inhibitory drive in GABAergic neurons•Human neuronal networks supported classification of three VUS as likely pathogenic

CACNA1A patient-derived human neuronal networks showed variant-specific alterations

A gain-of-function variant linked to epilepsy caused fragmented synchronization

This variant also led to an early increased inhibitory drive in GABAergic neurons

Human neuronal networks supported classification of three VUS as likely pathogenic

Van Bokhoven and colleagues demonstrate functional profiling of CACNA1A patient-derived neuronal networks, including some with CRISPR-Cas9-corrected/mimicked variants. They uncovered variant-specific alterations and provided support for classification of three variants of uncertain significance (VUS). Findings highlighted both the strengths and limitations of iPSC-based neuronal modelling, with significant implications for future endeavours to improve variant classification and understanding of CACNA1A-related disorders.