Introduction Alternating hemiplegia of childhood (AHC) is a rare and severe neurodevelopmental disorder characterized by recurrent hemiplegic episodes. Most AHC cases are sporadic and caused by ATP1A3 de novo mutations. This study aimed to search for the origin of ATP1A3 mutations in a Chinese AHC cohort.

Methods Sanger sequencing was performed in AHC patients. Micro-droplet digital PCR (mDDPCR) was applied for detecting mosaicism of the proband’s ATP1A3 mutations in the available families.

Results In 105 probands including 101 sporadic cases and four familial cases, 98 patients with ATP1A3 mutations, and 97.8% (91/93) was confirmed as de novo mutation. In blood samples, four asymptomatic cases with parental mosaicism, including two paternal and two maternal, and one mosaic proband with milder phenotype were identified. Sperm samples were collected from 51 fathers and multiple peripheral tissue samples were collected from 13 parents. Six (7.5%) parental mosaicisms were identified in multiple tissues, including four previously identified in blood, two additional cases identified from paternal sperm. Mosaicism identified in multiple tissues were with varied mutant allele fractions (MAFs, 0.03% - 33.03%).

Conclusion These results suggested that the MAF of mosaicism is related to the severity of phenotype. This study is the first systematic report of ATP1A3 mosaicism in AHC and demonstrated mosaicism as an under recognized source of previously considered “de novo” of AHC. The accurate quantification of mosaicism in ATP1A3 gene is important for the estimation of recurrence risk in the genetic counseling of AHC.