SHANK3 Deletion and Related Phenotypes in Chinese Children with Autism and shank3-KO Zebrafish Display Autistic-like Behaviours

Background:  Autism spectrum disorder (ASD) is well known as a heritable, debilitating neurodevelopmental disorder manifesting in early development. A mount of studies showed that SHANK3 gene had a strong causal relationship with ASD and/or 22q13.3 deletion syndrome. However, the data of Chinese ASD patients with SHANK3 deletion is insufficient and the mechanism is not clear.

Objectives:  N/A

Methods:  MLPA and Sanger sequencing were carried out to confirm the SHANK3 deficiency of Chinese children. Moreover, systematic and comprehensive evaluations were performed to Chinese-specific features. In addition, shank3 was knock-out (KO) using a CRISPR/Cas9 system in zebrafish to build a transgenic zebrafish model.

Results:  As to the patients, six participants lacked the whole gene of SHANK3 with 22q13.3 deletions ranging in size from 55 kb to 4.8 Mb and three participants with de novo SHANK3 mutation were included. They were characterized by high rates (100%) of ASD, developmental delay, hypotonia, several dysmorphologies and perception abreaction. New and rare features were also viewed in this study: ectropion of nostril sparse hair, ankle deformity, whole-body hairy, hanked-3-lap arms, snaggletoothed or extra teeth and unusual-dehydrated skin, and extreme hyperactivity/self-sitimulation. As to the zebrafish model, the shank3-KO zebrafish displayed varying degrees of developmental retardation compared with the wild-type zebrafish, such as ventral curled body, less melanin, less somites and so on. Moreover, the homozygous zebrafish were more significant than the heterozygotes. What’s more, in zebrafish social interaction test, shank3-KO zebrafish showed less interest exploring conspecific zebrafish both in swimming distance ratio and swimming time ratio. Furthermore, in zebrafish social preference test, shank3-KO zebrafish displayed reduced polarization of fish shoals, looser and larger schools, and higher percentage of fish leaving the group and spending time outside the shoal which implied a disorganized social structure. In addition to social deficits, the trace pattern analysis of zebrafish found several obvious behavioral stereotypies, such as repetitive, stereotypic “repeated self-rotation” swimming behavior.

Conclusions:  In our study, the severity of intellectual, hypotonia, and speech impairments were seen in SHANK3 deficiency which highlighted the prominence of SHANK3 in ASD. Zebrafish, a typical animal model, will play a critical role in further studying the relationship between phenotype and genotype of ASD and insighting into the molecular mechanisms underlying the clinical heterogeneity of ASD.