Transcriptome Analysis in Neuronal Cells of an Autistic Patient with 17p13.3 Duplication: Identification of Upregulation of Ywhae and Crk and Possible Contributor Factors for Penetrance.

Background:  Duplications in chromosomic region 17p13.3 have been identified in individuals with autism spectrum disorders (ASD) and intellectual disability (ID). However, 17p13.3 duplication carriers present incomplete penetrance and significant variability of the phenotype, suggesting that other genetic and/or environmental factors might be necessary for clinical manifestation. Association of genes in this region to ASD and ID, particularly YWHAE and CRK, is based on analysis of overlapping duplications and some sporadic functional studies showing the relevance of such genes to neurodevelopment, but no study has been conducted using the cells of the patients so far.

Objectives:  In this study, we aimed to investigate the consequences of 17p13.3 duplication for gene expression using neuronal cells derived from an autistic individual, as well as to explore other possible factors that might be contributing for the penetrance of the phenotype in this individual.

Methods:  Array-CGH was used to identify and delineate the boundaries of the duplication. Induced pluripotent stem cells (iPSC) were derived from stem cells from exfoliated teeth from the patient and 6 controls, and next differentiated in neuronal progenitor cells (NPCs) and neurons. We generated transcriptome data of these cells as well as exome data from DNA obtained from patient’s peripheral blood cells.

Results:  We have identified an autistic individual that harbors a de novo 345kb duplication in 17p13.3, spanning the genes ABR, BHLHA9, TUSC5, YWHAE, CRK and MYO1C. In accordance to phenotypic descriptions of other patients with duplications of similar size and location, the current case present only mild learning disabilities and mild autistic features. Our transcriptome analysis revealed 65 differentially expressed genes in patient’s NPCs compared to control NPCs, among which YWHAE and CRK was found as upregulated. We then searched for rare potential pathogenic variants in the remaining 63 differentially expressed genes, aiming to find other genetic alterations that could contribute for the penetrance of the phenotype. Interestingly, we identified a rare stop codon mutation in a downregulated gene, NGDN, which codifies an EIF4E-binding protein that regulates translation during nervous system development. Indeed, comparing genotypes found in exome and RNAseq data, we could identify a bias in the expression towards the normal allele, indicating the existence of RNA decay. Investigation of YWHAE, CRK and NGDN expression levels in the iPSC-derived neurons from the patient confirmed the same changes observed in NPCs.

Conclusions:

Our results add further support for the role of YWHAE and CRK for the manifestation of neurodevelopmental disorders presented by 17p13.3 duplication carriers. Also, we suggest that a loss of function mutation in NGDN may acts as the second hit necessary for the penetrance of ASD in the presence of 17p13.3 duplication.