To establish a minigene assay of splicing of the 3’ end of ATP1A3, enabling investigation of splice variants with varying neurological phenotypes.

Alternating hemiplegia in childhood (AHC) is a genetic disorder associated with severe paroxysmal and chronic neurological manifestations such as dystonia, plegia, ataxia, epilepsy, and intellectual disability. AHC is caused by heterozygous missense ATP1A3 variants that produce altered protein, likely with dominant-negative effects. Surprisingly, we identified a patient with AHC and an ATP1A3 NM_152296.5:c.2542+1G>A canonical splice variant that is predicted to be null, even though “null” variants that eliminate ATP1A3 expression can be asymptomatic. We hypothesized that this apparent contradiction could be explained if the variant RNA was not spliced as predicted, for example if it escaped NMD and produced an altered protein.

We cloned an ATP1A3 minigene expression construct containing the last 7 exons (17-23) and introduced the c.2542+1G>A variant in intron 18. Splicing effects were assessed by RT-PCR followed by Nanopore sequencing with the wildtype minigene as negative control.

The minigene displayed normal splicing of all exons and introns except intron 22, which was partially retained. Unexpectedly, the c.2542+1G>A variant caused retention of the first 124 bases of intron 18, rather than complete inclusion or exon-skipping. However, this consequence was still predicted to be null via NMD.

The ATP1A3 minigene is suitable for the investigation of splice variants in exons 17-22 and reveals an unexpected pattern of aberrant splicing associated with a severe neurological disease variant. Because minigene data cannot address whether the aberrantly spliced transcript undergoes NMD, complementing this data with RNA sequencing of patient biosamples will be important. Nevertheless, this minigene provides a platform for further comparison of splice variants associated with neurological disease against those in the general population, for which biosamples are often unavailable.