Cognitive impairment is a global challenge that creates cost, social, and economic challenges for society in many populations, in particular, older populations worldwide. Although, some effort has been made to understand the genes and biological pathways involved in cognitive functioning through gene expression analysis, there is still a lack of knowledge. Harries et al. (2012) performed gene expression analysis on 691 subjects from the InCHIANTI study (mean age 72.6 years) and reported that the CCR2 gene was associated with cognitive function (FDR = 0.077). Also, no significant pathways were identified. Nygaard et al. (2019) performed gene expression analysis on 235 monozygotic (MZ) twin pairs and reported POU6F1 gene (FDR = 0.09) and significant pathways such as protein metabolism, translation, RNA metabolism, infectious disease, and the immune system.

The popular statistical models used in the analysis of gene expression data are usually linear models, which are controlled by multiple assumptions, including normality of phenotype and linear relations between expression level and the phenotype. In the case of having twins in the data, linear mixed-effect models are appropriate to deal with the correlation structure in the data. Imposing multiple assumptions in linear models might be the reason for having a smaller number of important markers in gene expression analysis. However, recently a couple of studies have shown the strength of generalize correlation coefficient (GCC) as a non-parametric method that is able to identify different patterns, deal with correlated twin samples as well as non-normality of the phenotype without imposing strict assumptions (Reshef et al., 2011; Speed, 2011; Murrell et al., 2016; Tan et al., 2017; Mohammadnejad et al., 2020, 2021). In fact, linear models are well-designed when there is a perfect linear relation; hence we applied both GCC and linear models.

Transcription factors (TFs) are specific DNA sequences that affect gene expression by promoting or repressing the target genes. Mutation in TFs and TF binding sites determine many human diseases. The group of genes that are controlled by TFs are called regulons (Lambert et al., 2018).

This study aimed to perform two analyses: (1) a global gene expression analysis of cognitive function measured in monozygotic (MZ) twins to identify significant genes and pathways associated with the phenotype by applying the assumption-free GCC and linear models, (2) investigate the significance of previously reported cognitive function-related TFs through a gene regulatory network analysis.

Through applying both the assumption-free GCC and linear models, this exploratory study was able to capture diverse patterns of relations not limited to those from linear models. We were able to identify interesting genes and pathways implicated in cognitive function. Also, the novel transcription regulatory analysis paved the way for the detection of significant regulons associated with cognitive function.

The APOBEC3G gene belongs to a family of proteins grouped together due to their homology with the cytidine deaminase APOBEC1 (https://en.wikipedia.org/wiki/APOBEC3G). A very recent paper by Smith et al. (2020) reported that the APOBEC1 gene affected cognitive aging in animals. In addition, Cole et al. (2017) have reported that APOBEC1 is an essential regulatory mechanism of microglia (MG) function and is critical for overall brain homeostasis and healthy aging. The other gene SLC45A1, is highly expressed in the brain (https://gtexportal.org/home/). It is a protein-coding gene and associated with intellectual developmental disorder with neuropsychiatric features and autosomal recessive non-syndromic intellectual disability (https://www.genecards.org/cgi-bin/carddisp.pl?gene=SLC45A1). Srour et al. (2017) studied homozygous missense variants in SLC45A1 on four affected children from two unrelated consanguineous families with moderate to severe intellectual disability associated with epilepsy and variable neuropsychiatric features. They concluded that autosomal-recessive mutations in SLC45A1 result in intellectual disability, movement disorder, and epilepsy. SLC45A1 is thus the second cerebral glucose transporter, in addition to GLUT1, to be involved in human disease and implicated in neurodevelopmental disability. Also, SLC proteins are of great importance in the elucidation of neurodegenerative disorder mechanisms due to their important role in the synaptic regulation of neurotransmitters (Aykaç and Sehirli, 2020). Martínez-García and colleagues studied the association of rs6688832 and rs34603401 polymorphisms in the H6PD gene with obesity and polycystic ovary syndrome (PCOS) on 237 cases and 135 controls and they found that the variants in the H6PD gene were associated with obesity and PCOS (Martínez-García et al., 2012).

The other gene is PDE4D which is already reported to encode a cyclic AMP (cAMP) regulator which places PDE4D-related acrodysostosis in the same family of diseases as pseudohypoparathyroidism, pseudohypoparathyroidism, PRKAR1A-related acrodysostosis, and brachydactyly mental retardation syndrome, which are all characterized by cognitive impairment and short distal extremities (Lynch et al., 2013). Also, using gene knock-out and miRNA-induced gene knock-down in mice, the PDE4D gene and particularly its long-form isoforms has been shown to play a vital role in the mediation of memory and hippocampal neurogenesis which is mediated by cAMP signaling (Li et al., 2011; Michot et al., 2012). The gene GRIN3B is associated with depersonalization disorder and schizophrenia (https://www.genecards.org/cgi-bin/carddisp.pl?gene=GRIN3B). Additionally, in a study, Hornig et al. performed whole exome-sequencing in eight family members with a strong history of psychotic disorders over three generations and they found in all the affected family members frameshift mutation rs10666583 in the GRIN3B gene, which codes for the GluN3B subunit of the NMDA receptor (Hornig et al., 2017). NMDA receptors are ligand-gated cation channels that are blocked in the resting state by magnesium ions. They are involved in learning and memory, synaptic plasticity, and synaptogenesis (Harris et al., 1984; Normann et al., 2000; Lynch, 2004; Normann and Clark, 2005; Fan et al., 2014).

The PIGC gene is associated with diseases including glycosylphosphatidylinositol biosynthesis defect 16 and autosomal recessive non-syndromic intellectual disability (https://www.genecards.org/cgi-bin/carddisp.pl?gene=PIGC) which are both linked to intellectual disabilities. Moreover, Edvardson and colleagues studied to find disease-causing mutations in three patients from two unrelated families with severe intellectual disability, global developmental delay, and drug-responsive seizure disorder. They concluded that mutations in the PIGC gene were associated with epilepsy and intellectual disability (Edvardson et al., 2017).

We found interesting and important pathways which might be implicated in cognitive impairment (Table 2). Ding et al. (2005) reported that in patients with mild cognitive impairment and Alzheimer's disease (AD), there is significant dysfunction in ribosome function that is not observed in the cerebellum of the same patients. Ribosome dysfunction is associated with a decline rate in protein synthesis, ribosomal RNA and tRNA levels, and increased RNA oxidation. Focal adhesion involves the integration of the adhesion, the communication between the extracellular matrix and the actin cytoskeleton, and the regulation of many cell types. Loss of cell adhesion can lead to cell death and altered focal signaling has been associated with synaptic loss, which may cause AD (Caltagarone et al., 2007). Wilson et al. (2002) reported that with increasing age peripheral cytokine dysregulation interacts with cognitive aging. Magaki et al. (2007) showed that alteration in cytokines through peripheral blood mononuclear cells (PBMCs) might be detected early in mild cognitive impairment. Additionally, other studies have been done on the role of cytokines in AD, cognitive impairment, and neurological disorders (Aarli, 2005; Nagae and Araki, 2016). Chang et al. (2012) discussed that p53 interacts with cellular factors, viral factors, and small RNAs, explaining its role in the development of neurodegenerative diseases. Previous studies point to evidence of the role and pathogenesis of Huntington's disease, endocytosis, eukaryotic translation elongation, nervous system development, influenza infection, metabolism of RNA, and cell cycle in relation to cognitive impairment, AD, and neurological diseases (Peavy et al., 2010; Jurgens et al., 2012; Mufson et al., 2012; Beckelman et al., 2016; Barbash et al., 2017; Zhu et al., 2020).

A total number of five significant regulons were enriched by GSEA-2T analysis of transcriptional regulation, in which CTCF and REST were identified as activated regulons and SP3, SRF, and XBP1 as repressed regulons in our study. A gene expression study on animal adult forebrain-restricted SRF deletion reported the decreased expression of several gene-containing serum response elements (SRE). And they proposed that these deficits in gene expression indicate SRF's role in the induction of genes necessary for long-term memory formation and the late phase of long-term potentiation-like (LTP) plasticity (Ramanan et al., 2005; Etkin et al., 2006). In our study, we also found the decreased expression of TF genes included in the SRF regulon with cognitive function. Yamakawa et al. (2017) discussed that SP3 tends to be a major negative regulator of synaptic gene expression and synaptic activity, which is also likely to play a significant role in cognitive decline in AD patients. They reported that SP3 and histone deacetylase HDAC2 negatively regulate synaptic function in neurons. Our study showed the downregulation of the SP3 regulon with cognitive function. Some studies have shown the role of XBP1 in endoplasmic reticulum stress, memory, and cognition (Valdés et al., 2014; Cissé et al., 2017a), rescuing hippocampal synaptic plasticity and memory through activating the Kalirin-7 (Kal7) pathway (Cissé et al., 2017b), XBP1's expression corrected age-associated changes in synaptic function (Cabral-Miranda et al., 2020). The other TF, CTCF has been studied by Choi et al. (2021) in adult CTCF cKO mice and concluded that deficiency in CTCF results in cognitive deficits. Some studies have reported the importance of REST due to its polymorphism role in cognitive function and the activation state of REST in the aging brain, which may differentiate neuroprotection from neurodegeneration (Lu et al., 2014; Warburton et al., 2016).

Overall, through applying GCC as a complementary method along with the linear models, this exploratory study was able to detect more important and meaningful differentially expressed genes and biological pathways implicated in cognitive function. Additionally, applying transcriptional analysis could reveal the link between significant regulons and cognition which further confirms that previously noted TFs are associated with cognitive function.

According to Danish and EU legislations, transfer and sharing of individual-level data require prior approval from the Danish Data Protection Agency and require that data sharing requests are dealt with on a case-by-case basis. However, we welcome any enquiries regarding collaboration and individual requests for data sharing. Requests can be directed to JH, jhjelmborg@health.sdu.dk.

The studies involving human participants were reviewed and approved by The Regional Scientific Ethical Committees for Southern Denmark (S-VF-19980072). The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

JH and QT contributed to the conception and design. AM performed the data analysis and wrote the manuscript. All authors read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.