Differential expression analysis of the ZNF584 gene in children with autism spectrum disorder

Introduction: Autism spectrum disorder (ASD) is a heterogeneous set of neurodevelopmental conditions affecting social communication in young children and causing restricted repetitive behavior [1]. ASD is highly heritable, although only a small number of autism cases have been associated with known single-nucleotide polymorphisms, copy number variants, or large chromosomal aberrations [2]. Alterations in gene expression in the peripheral blood of affected individuals have also been well-documented [2]. In this regard, zinc finger (ZNF) proteins are ubiquitously expressed in various cell types and play central roles in gene regulation. Furthermore, studies have underlined the involvement of ZNF proteins in neuropsychiatric disorders [3].

Purpose: This study aims to explore the expression of ZNF584 at the transcriptional level in peripheral blood samples of children with ASD in comparison with typically developing children (TDC) of the control group.

Methods: This study enlists 23 children with ASD and 21 TDC of the control group. All ASD diagnoses were established by certified professionals according to internationally accepted criteria, including CARS, GARS, ADOS, and DSM-5.

Total RNA samples from all participants were purified using a PAXgene kit (PreAnalytiX). After a DNase I treatment (Promega), a total amount of 1 µg RNA was reverse-transcribed using Maxima reverse transcriptase (Thermo Scientific). Quantitative PCR analyses were performed in an ABI 7500 thermal cycler with a two-step protocol and Maxima SYBR Green chemistry (Thermo Scientific).

The obtained individual expression profiles were tested for significance using a non-parametric Mann-Whitney U test, and their discriminative potential was further evaluated using a receiver operating characteristic (ROC) analysis (pROC package version 1.18.5, R version 4.3.2)

Results: The data from the relative quantitation RT-qPCR experiments demonstrated statistically significant differential expression of the ZNF584 gene at the transcriptional level between the ASD and TDC groups. According to the applied Mann-Whitney U test, the obtained delta-Ct values for the gene of interest were higher in the TDC group (Median = 14.066) than in the ASD group (Median = 15.106): U = 103, z = -3.09, p = 0.002. In terms of fold-change differences, these discrepancies in delta-Ct data among groups translate into a two-fold upregulation of ZNF584 in the ASD group compared to the control group. The subsequent ROC analysis of the ZNF584 differential expression confirmed its discriminative potential to correctly stratify the participants from the two examined groups: AUC = 0.7761 (95% CI [0.627-0.924] (DeLong), p < 0.001); Sensitivity: 0.73, Specificity: 0.70.

Discussion: To our knowledge, this is the first study to suggest differential expression of ZNF584 at the transcriptional level in the peripheral blood of children with ASD. Intriguingly, ZNF584 has been examined in the context of human intelligence evolution and cognitive function [4,5]. Moreover, our results are concordant with a previous study reporting differential expression of several other ZNF factors in the peripheral blood of adult ASD patients [2]. Further mechanistic exploration is required for the complete elucidation of the role of ZNF584 in ASD.

Conclusion: Based on our experimental data, we can suggest a significant upregulation of the ZNF584 gene in children with ASD.

References

1. American Psychiatric Association, 2013. Diagnostic and statistical manual of mental disorders, fifth ed. American Psychiatric Association Publishing, Washington.
2. Kong, S.W., Collins, C.D., Shimizu-Motohashi, Y., Holm, I.A., Campbell, M.G., Lee, I.H., Brewster, S.J., Hanson, E., Harris, H.K., Lowe, K.R., Saada, A., Mora, A., Madison, K., Hundley, R., Egan, J., McCarthy, J., Eran, A., Galdzicki, M., Rappaport, L., et al., 2012. Characteristics and predictive value of blood transcriptome signature in males with autism spectrum disorders. PLoS One 7 (12), e49475.
3. Bu, S., Lv, Y., Liu, Y., Qiao, S., Wang, H., 2021. Zinc Finger Proteins in Neuro-Related Diseases Progression. Front. Neurosci 15, 760567.
4. Li, M., Zhang, W., Zhou, X., 2020. Identification of genes involved in the evolution of human intelligence through combination of inter-species and intra-species genetic variations. PeerJ 8, e8912.
5. Savage, J.E., Jansen, P.R., Stringer, S., Watanabe, K., Bryois, J., de Leeuw, C.A., Nagel, M., Awasthi, S.., Barr, P.B., Coleman, J.R.I., Grasby, K.L., Hammerschlag, A.R., Kaminski, J.A., Karlsson, R., Krapohl, E., Lam, M., Nygaard, M., Reynolds, C.A., Trampush, J.W., et al., 2018. Genome-wide association meta-analysis in 269,867 individuals identifies new genetic and functional links to intelligence. Nat. Genet 50 (7), 912-919. The authors declare no conflicts of interest. This study was supported by the Bulgarian National Science Fund, Grant No. КП-06-М91/5 (BG-175467353-2025-07-0019-C01): Comparative analysis of inflammatory factors in individuals with overweight and obesity.