Based on data coming from several animal and cellular studies that highlight the molecular links between NRN1, BDNF and CACNA1C genes (Fujino et al., 2003; Wibrand et al., 2006), this study explores the neurobiological pathways through which the epistasis between these synaptic plasticity-related genes may be associated with the clinical presentation of SZ. By combining genetic, neuroimaging and clinical data, the main findings show the epistasis between NRN1 and its interactors (BDNF and CACNA1C) on SZ clinical features and indicate that this joint genetic background may exert its effect through the impact on neuroanatomical measures.
Regarding our analyses exploring the epistatic effect on the risk for SZ, we did not detect any significant association. While the individual effect of NRN1, BDNF and CACNA1C on the risk for SZ is reported in different studies, there is little evidence showing that NRN1 role seems to be not independent of BDNF (Fatjó-Vilas et al., 2016; Prats et al., 2017), and no previous study has explored the epistasis with CACNA1C. Thus, the counterpart to the novelty in our study is the challenge in comparability, as there is only one prior study (Fatjó-Vilas et al., 2016) that described a significant interaction effect between NRN1-rs9379002 and BDNF-rs6265 on a broader clinical spectrum, including both SZ and bipolar disorder. These discrepancies could be due to diagnosis and sample size differences. Nonetheless, the current results do not indicate a direct epistatic effect on the risk of SZ while revealing significant epistatic effects relevant to the clinical presentation of the disorder.
In this sense, we found a significant interaction between NRN1-rs10484320 and BDNF-rs6265 impacting PANSS general psychopathology, which encompasses symptoms like anxiety, guilt, tension, depression, and disorientation (Kay et al., 1987). Albeit earlier studies noted the negative impact of BDNF-rs6265 ValVal genotype on various PANSS subscales (Chang et al., 2009; Numata et al., 2006; Zhai et al., 2013), our data revealed that NRN1-rs10484320 modifies this effect. We observed more severe symptoms in patients with the T allele and the ValVal genotype and those with the CC genotype when they also had the Met allele. We also identified a significant interaction between NRN1-rs4960155 and CACNA1C-rs1006737 affecting GAF scores, summarising personal, social, and psychological functioning (Endicott et al., 1976). Again, previous research has established the CACNA1C-rs1006737 A allele as a risk factor for SZ through GWAS and meta-analytic approaches (Liu et al., 2020; Trubetskoy et al., 2022) and has demonstrated its detrimental effect on longitudinal GAF scores and recovery after psychotic episodes (Heilbronner et al., 2015). Our data adds to such evidence by revealing that NRN1-rs4960155 modulates the CACNA1C effects. Patients with TT genotype and the A allele for CACNA1C and those with the GG genotype but carrying the C allele for CACNA1C, exhibited poorer functioning. It is worth mentioning that both NRN1 SNPs, rs10484320 and rs4960155, have been previously associated with the risk of SZ-spectrum disorders and have been found to influence IQ scores among these individuals (Chandler et al., 2010; Fatjó-Vilas et al., 2016).
Nevertheless, taking into account the clinical heterogeneity of SZ and its complex aetiology, expecting a direct influence of genetic background on the disorder's clinical presentation seems unrealistic; instead, genetic networks might modulate a lower-level trait, which, in turn, sustains the manifestation of symptoms (Glahn et al., 2010; Meijer et al., 2021). Therefore, following other previous studies (Kirschner et al., 2020; Miranda et al., 2019; Sudre et al., 2020), we have proposed that deconstructing SZ into biologically validated and stable trait markers, such as brain structural measures and investigating their role in mediating symptomatology could help to fill the gap in the path from synaptic plasticity genetic variability to the complex and heterogeneous clinical presentation of the disorder.
In this regard, we discovered that NRN1-rs2208870 and several upstream variants (rs10484320, rs4960155, rs9379002, rs1475157) interact with BDNF-rs6265 to modulate CSA and CV among individuals with SZ. Additionally, we identified a singular interaction between NRN1-rs1475157 and CACNA1C-rs106737 that influences CSA. These findings highlight the role of epistatic interactions involving NRN1, BDNF, and CACNA1C in contributing to the strong genetic underpinnings of CSA, which is estimated to have a heritability of 91% (Eyler et al., 2012). The critical roles of these genes in neural development (Sasi et al., 2017; J. Yao et al., 2018) also support its inclusion in the pool of genetic factors influencing CSA, which is driven by regulatory elements active during prenatal cortical development (Grasby et al., 2020).
Those brain regions significantly modulated by epistasis effects, mainly frontal regions as well as some parietal and temporal regions, have been previously reported to present CSA and CV reductions in SZ patients (Erp et al., 2018; Madre et al., 2020; Rimol et al., 2012). Evidence from voxel-based morphometry meta-analyses has also evidenced CV reductions, especially in frontotemporal regions (Bora et al., 2011; Honea et al., 2005). Though our analysis was confined to SZ patients, our findings indicate that these specific epistatic combinations distinctly impact brain structure in SZ patients. Thus, it is plausible that these genetic interactions might also play a role in the molecular mechanisms contributing to the previously mentioned differences in cortical structure between SZ and HS.
From all the epistasis effects on brain structure, we highlight the NRN1-rs10484320 x BDNF-rs6265 interaction effect on the L-LOFC volume since these genetic variants also jointly modulate PANSS general psychopathology. Our moderated mediation model was designed to deep into the relationship between these significant associations and confirmed the mediation effect of the L-LOFC in the relationship between the NRN1-rs10484320 x BDNF-rs6265 epistasis and the PANSS general psychopathology scores.
However, contrary to the expected, the same patients who presented the lowest PANSS general psychopathology scores, those carriers of the CC genotype for NRN1-rs10484320 and the ValVal genotype for BDNF-rs6265, were the ones with the smaller left L-LOFC volume. The L-LOFC, as part of a functional network including the medial prefrontal cortex (Öngür & Price, 2000), plays multiple roles, such as the integration of multiple sensory information, modulation of visceral reactions, and participation in learning, prediction, and decision-making for emotional and reward-related behaviours (Kringelbach & Rolls, 2004; Rolls, 2004). Findings in SZ regarding structural alterations of the OFC are controversial. While some studies have reported volume reductions (Madre et al., 2020; Rimol et al., 2012), others have described increased volume of the left OFC (Lacerda et al., 2007). These incongruities are not clarified by exploring the relationship with symptoms, as both volume increase and decrease have been associated with SZ symptomatology (Baaré et al., 1999; Gur et al., 2000; Koutsouleris et al., 2008; Lacerda et al., 2007; Nakamura et al., 2008). Interestingly, proteomic studies have linked N-methyl-D-aspartate (NMDA) receptors hypofunction and disruption of calcium homeostasis with OFC volumetric alterations in SZ (Nascimento & Martins-de-Souza, 2015; Velásquez et al., 2019). At the clinical level, decreasing glutamatergic neurotransmission through NMDA receptor antagonists generates and worsens psychotic traits, suggesting its significance as a major pathway for symptom development in SZ (Lewis and González-Burgos, 2007). In fact, NRN1 expression in the cortex is regulated by Ca2+ signalling via the NMDA receptor (Fujino et al., 2003), and NMDA receptor-mediated neurotransmission and plasticity are particularly affected by BDNF-rs6265 genotype within the hippocampus and infralimbic medial prefrontal cortex (Ninan et al., 2010; Pattwell et al., 2012). A significant pathway in the development of positive and negative symptoms may result from the reduced activity of glutamate, mainly mediated through the NMDA receptor (Lewis and González-Burgos, 2007). Therefore, both neurotrophic factors might be taking part in the molecular pathways underlying the volumetric differences of the L-LOFC in SZ subjects that are behaviourally reflected in general psychopathology. Thus, our findings offer insight into the previously debated results, providing evidence that these genetic factors may contribute to volumetric variability among individuals, which, in turn, may underlie the emergence of general psychopathology during lifespan.
To understand how those genetic variants modulate psychopathology through their impact on brain structure, the functional consequences of those polymorphisms must be considered. On the one hand, it is worth noting that the BDNF-rs6265 Met variant has been demonstrated to affect both its intracellular distribution and activity-dependent secretion (Chen et al., 2005, 2006; Chiaruttini et al., 2009; Egan et al., 2003). On the other hand, it should be contemplated that the NRN1-rs10484320 has been described by the GTEx Project to impact NRN1 expression on the hippocampus. Therefore, although our reported statistical interaction does not directly imply a biological interaction, it is plausible to hypothesise that both polymorphisms could influence the brain availability or function of NRN1.
Finally, we should account for some limitations of this study. First, the sample could be relatively small. Nevertheless, we concurrently detected the interaction effect between NRN1-rs10484320 and BDNF-rs6265 on SZ symptomatology using two distinct methods: firstly, through an association analysis following multiple testing correction, and secondly, by employing a brain imaging approach with a rigorous significance threshold in conjunction with the cluster-wise correction approach. Also, while the present study has not directly examined the molecular mechanism through which these molecular interactions might contribute to the clinical presentation of SZ, our findings, jointly with existing functional data, underscore the necessity for cell-based investigations that incorporate genetic variability information. Lastly, although the homogeneity of our sample concerning ethnicity and demographic variables minimises the potential for error, it also limits the extrapolation of our results. It demands new studies in larger samples with equal representation of those populations.