Schizophrenia is a complex mental illness that usually begins in young adulthood and profoundly affects an individual's intellectual, emotional, and social functioning. Schizophrenia is characterized by positive symptoms such as hallucinations, delusions, disorganized speech, and disorganized behavior, as well as negative symptoms such as a lack of motivation, social withdrawal, and reduced emotional expression. Although positive and negative symptoms come to the fore in a descriptive sense, cognitive symptoms are one of the core features of schizophrenia and negatively affect the daily life, functioning, and social life of people with schizophrenia (Guzman and Harvey, 2024; Vita et al., 2024; Green et al., 2019). Cognitive impairments observed in schizophrenia cover a wide range, from perceiving, processing, and responding to cues in the social environment to difficulties with attention, memory processes, processing speed, reasoning and problem-solving (Green et al., 2019).

Cognitive impairment in schizophrenia is an important area of concern and research in both treatment-responsive and treatment-resistant patients. Treatment-resistant schizophrenia (TRS) is defined as the inability to respond to at least two non-clozapine antipsychotic treatments despite adequate dose, duration, and adherence (Elkis, 2007; Kane et al., 2019; Buckley 2020). Approximately one-third of schizophrenia patients are treatment-resistant (Howes et al. 2017). Clozapine is the standard treatment approved for TRS and recommended in various schizophrenia treatment guidelines (Barnes et al., 2020; Keepers et al., 2020; Correll, 2022). Although current approaches draw attention to the efficacy of other second-generation antipsychotics such as olanzapine, clozapine has an important place in the treatment of TRS (Efthimiou et al., 2024; Schneider-Toma et al., 2025). Clozapine is also effective in preventing suicide and aggression in patients with schizophrenia (Masdrakis and Baldwin, 2023), and based on data from the International Suicide Prevention Trial (InterSePT) (Meltzer et al., 2003), the Food and Drug Administration (FDA) approved clozapine for the treatment of symptoms of suicidality in patients with schizophrenia/schizoaffective disorder. Despite the efficacy of clozapine in TRS and evidence that patients on clozapine have lower mortality and better quality of life (Lee et al., 2023), many psychiatrists are reluctant to prescribe clozapine because of serious side effects such as agranulocytosis, decreased epileptic threshold, myocarditis, ileus, and hypersalivation-induced pneumonia (Smessaert et al., 2024). Although these side effects of clozapine are rare, clinicians' fears lead to inappropriate treatment approaches such as multiple antipsychotic use, high-dose antipsychotic trials, and an average delay of four years in initiating clozapine, even in cases where a switch to clozapine is recommended by guidelines (Howes et al. 2012).

Although clozapine is an important agent in the treatment of TRS, data on its effect on the cognitive symptoms of schizophrenia are conflicting. In a meta-analysis evaluating the efficacy of antipsychotics on cognitive performance in people with psychotic disorders, clozapine treatment was associated with poorer performance on tasks related to executive function, verbal learning, and visual configuration tests (Baldez et al., 2021). In another study, haloperidol, fluphenazine, and clozapine were associated with poorer performance in several cognitive domains, including verbal learning, working memory, visual learning, processing speed, and social cognition (Feber et al., 2025). The results of a recent meta-analysis suggest that the use of clozapine for six months or longer in TRS is associated with improvement in cognitive performance; patients with younger age, higher education levels, and improvement in positive symptoms are more likely to improve their cognitive performance (Cheuk et al., 2024). Differences between studies may be due to heterogeneity in the assessment of treatment resistance and worse cognitive performance in the TRS group. A study comparing the cognitive performance of TRS patients treated with clozapine and patients responding to non-clozapine oral antipsychotics found that TRS patients performed worse (Suzuki et al., 2024).

One of the issues investigated in the study of the effects of clozapine on cognitive performance is the effect of plasma clozapine (CLZ), plasma N-desmethylclozapine (NDMC) levels, and the CLZ/NDMC ratio on cognitive performance. Anticholinergic activity is associated with deterioration in cognitive performance tests (Mancini et al., 2025). The agonistic effect on muscarinic receptors could theoretically have a beneficial effect on cognitive symptoms in schizophrenia. Indeed, this is one of the reasons that led to the development of the M1/M4 agonist KARTXs for the treatment of schizophrenia (Ramey and Silva Almodóvar, 2025). M1 and M4 receptors can reduce the severity of positive, negative, and cognitive symptoms of the disorder (Dean et al., 2023). While clozapine itself is antagonistic at M1 receptors, its active metabolite N-desmethylclozapine is agonistic (Rajji et al., 2015). Clozapine also has a partial agonistic effect on M4 receptors (Morrison et al., 2025). Given all these properties, it has been hypothesized that CLZ, NDMC levels, and the CLZ/NDMC ratio may be related to cognitive performance in TRS. Rajji et al. (2015) found that the CLZ/NDMC ratio was significantly and negatively associated with working memory performance. They did not report a direct relationship between CLZ or NDMC concentrations and working memory performance. In another study, the CLZ/NDMC ratio was found to be associated with the severity of cognitive impairment in patients with TRS (Suzuki et al., 2024). Another recent study found a negative causal relationship between the CLZ/NDMC ratio and attention/vigilance, and no significant correlation between the CLZ/NDMC ratio and processing speed, reasoning/problem solving, working memory, verbal learning, or visual learning (Moscou et al., 2024). Arnautovska et al. (2023) also reported no correlation between CLZ/NDMC ratio and verbal and visual learning.

The effect of M1 receptor polymorphisms on cognitive symptoms in schizophrenia has also been investigated. The gene CHRM1, located on chromosome 11q12.3, encodes the M1 receptor. There are fifteen single nucleotide polymorphisms (SNPs) in this gene region, one of which is known as C267A (rs2067477, https://www.ncbi.nlm.nih.gov/snp/rs2067477). The C267A SNP is a silent mutation that does not change the protein sequence. It involves the conversion of cytosine (C) to adenine (A) at position 267 in the gene region (Brann et al., 1993). Liao et al. (2003) and Scarr et al. (2012) found that individuals with the heterozygous genotype (267C/A) showed better cognitive performance related to prefrontal cortical function than those with the homozygous genotype (267C/C) on the Wisconsin Card Sorting Test (WCST). However, Carruthers et al. (2019) found no significant difference in performance between the two groups during the Wechsler Test of Adult Reading (WTAR) task. Nevertheless, the effect of the clozapine-M1 receptor polymorphism interaction on cognitive performance has not been sufficiently studied. Kır et al. (2020) reported no significant difference in performance between individuals with and without genetic differences on the N-back task in TRS patients taking clozapine. Therefore, further research is needed to clarify the potential interaction between clozapine treatment and M1 receptor polymorphisms in influencing cognitive outcomes in TRS.

Existing research on TRS has revealed disruptions in both resting-state and task-based functional connectivity patterns. Resting-state studies consistently show reduced connectivity within the default mode network (DMN) and impaired anti-correlation between the DMN and task-positive networks, such as the frontoparietal network (FPN) and salience network (SN) (Cheuk et al., 2024; Pankow et al., 2015; Repovš and Barch, 2012). These abnormalities have been linked to deficits in self-referential processes, cognitive control, and attentional regulation (Pankow et al., 2015; Wu and Jiang, 2020). Task-based studies highlight that TRS patients struggle to transition effectively from DMN-dominant resting states to task-positive activation, reflecting delayed or incomplete engagement of task-relevant networks during cognitive tasks, such as working memory (Godwin et al., 2017; Repovš and Barch, 2012; Sheffield and Barch, 2016). While TRS patients often exhibit hypo-connectivity in core networks like the FPN, some compensate through hyperconnectivity in auxiliary regions (e.g., anterior cingulate cortex, temporal areas), which may sustain task performance but at a higher metabolic cost (Pang et al., 2023; Wu and Jiang, 2020). Clozapine, the standard treatment for TRS, can modulate these patterns, enhancing focal activations in task-relevant regions and partially normalizing connectivity, although its effects vary based on genetic and neurobiological factors (Cheuk et al., 2024; Wu and Jiang, 2020; Wolf et al., 2011).

The current study aims to investigate the effect of the M1 cholinergic receptor polymorphism (rs2067477) as a potential factor influencing clozapine’s effects on functional connectivity patterns in patients with treatment-resistant schizophrenia (TRS) receiving clozapine monotherapy. Although several studies have examined both resting-state and task-induced functional connectivity in TRS patients, to the best of our knowledge, the potential influence of the rs2067477 polymorphism on task-induced functional connectivity has not yet been investigated. The M1 receptor polymorphism is thought to modulate clozapine’s effects on brain activity and connectivity. We analyzed functional connectivity patterns using a network perspective, examining connections between cortical regions during N-back working memory task performance. This approach is particularly valuable in psychiatric populations, as cognitive functions such as working memory are supported by coordinated activity across distributed neural networks rather than isolated brain regions. The Wavelet Transform Coherence (WTC) method was applied to functional near-infrared spectroscopy (fNIRS) data to compare brain connectivity patterns between wild-type (CC) and non-wild-type (CA/AA) rs2067477 genotype groups. In addition, graph-based analysis was conducted to further explore differences at the network level. Our primary hypothesis is that patients with the wild-type genotype (CC) will exhibit stronger and more integrated functional connectivity in frontoparietal networks during working memory tasks compared to non-wild-type (CA/AA) individuals, reflecting a potential modulatory effect of the rs2067477 polymorphism on cognition-related neural networks in schizophrenia.