INTRODUCTION
Schizophrenia is a severely disabling psychiatric disease characterized by complex clinical features and a latent period of onset and development, making early judgment and intervention challenging before typical psychiatric symptoms emerge. The diagnosis and treatment of schizophrenia depend on the disease course, including physiological function decline and disability progression [1]. Studies have indicated that early intervention can improve prognosis, enhance social recovery, improve quality of life, and reduce social burdens [2-4].
Currently, the diagnosis of schizophrenia and related spectrum disorders occurs after the appearance of psychiatric symptoms, making early-stage diagnosis difficult [5]. Research indicates that schizophrenia integrates genetic information and neuromechanistic disorders. Abnormalities can impair the analytical ability and integrity of neural circuits and decrease neuron synapse function [6,7], which are considered risk factors in disease development. The earliest studies on high-risk samples for schizophrenia originated in the 1960s [8]. This high-risk population is characterized by familial genetic factors or a preclinical phase insufficient for diagnosis [9].
Schizophrenia progresses through stages. High-risk individuals with a positive family history carry more susceptible genes, known as a “schizotypic trait.” Influenced by environmental factors, these individuals may develop schizoid disorder with typical mental and behavioral changes, also known as the prodromal stage or clinical high-risk stage of schizophrenia [10]. At this stage, psychiatric symptoms and social function alterations contribute to the onset of schizophrenia, as subsequent research elaborated on schizophrenia development [11]. By improving the theory of the schizotypic trait, endophenotype-related studies were promoted, providing a clearer understanding of schizophrenia.
Generally, schizophrenia consists of three progressive stages: the latent stage (the “schizotypic trait,” usually accompanied by social function changes, negative symptoms, and cognitive damage), the prodromal stage (also called the “super highrisk stage,” with clinical high-risk state and functional degradation), and the clinical diagnosis stage (definitive diagnosis of schizophrenia) [12].
Studies on confirmed patients and high-risk populations showed that various typical disorders, including thought, social function, sensory perception, and cognition, may be common in the super high-risk stage. The 2-year progression risk is 30%-40% [13,14]. Difficulties in psychopathology judgment can affect early intervention [9]. Therefore, recognizing this population can decrease onset risk and improve prognosis [15].
Research on high-risk populations includes genetic factors and clinical features, similar to case-control studies of schizophrenia. Individuals with a positive family history of schizophrenia in first-degree relatives are described as having genetic high risk (GHR) [16]. The GHR population may have more genetic predisposition factors. Clinical high risk (CHR) individuals exhibit greater clinical alterations in thoughts, perception, and cognition [17].
Previously, we conducted research on the effects of MTHFR in schizophrenic patients [18,19]. From genetic and symptomatic perspectives, it was reported that gene polymorphism is related to disease progression in cardiovascular and metabolic disorders [20,21]. We then considered whether MTHFR plays a role in the high-risk stage of schizophrenia or if there is altered gene polymorphism across different stages of the disease. By including CHR, GHR, and healthy controls (CONs), we analyzed the effects of MTHFR on the risk progression of schizophrenia, hypothesizing that high polymorphism of this gene would promote the progression of schizophrenia.
METHODS
Participants and symptom ratings
A total of 163 individuals were enrolled through the outpatient unit of Beijing Tongren Hospital. Structured Interview for Psychosis Risk Syndromes (SIPS) was used to screen the CHR individuals. Meanwhile, the first-degree relatives of schizophrenic patients were randomly enrolled as the GHR group. All potential diagnoses of mental or somatic-related disorders were excluded. The study included 76 healthy CONs, 31 GHR individuals (first-degree relatives of schizophrenic patients), and 56 CHR individuals (SIPS verified), confirmed by a consensus medical conference based on clinical interviews, chart reviews, and clinical history reviews with treating physicians. For some analyses, the GHR and CHR groups were merged into one high-risk (HR) group consisting of 87 individuals. On account of preclinical features, both groups maintain several susceptible genes that may promote higher psychosis risk. Beyond that, the positive family history in GHR deemed to be a subclinical symptom, is also a justification for merging. All groups completed the MATRICS Consensus Cognitive Battery (MCCB) to assess cognitive ability, administered by trained raters who were blind to the groups and genotypes. Other recorded clinical features included gender, age, family history, cognitive scores, and SIPS scores. Informed consent was obtained from all individual participants included in the study. This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Beijing Tongren Hospital (Ethical approval number: 2022103FS-2).
Peripheral samples
Peripheral blood samples were collected from the departments of Beijing Tongren Hospital. Apheresis was conducted to obtain white blood cells, from which DNA samples were extracted and stored at -80°C for genotype assays.
MTHFR genotype assay
Three single nucleotide polymorphisms (SNPs) on MTHFR were detected using Taqman fluorescence probes in the ABI PRISM 7500 Sequence Detection System (Applied Biosystems). The primers and probes were commercial products from Thermo Fisher Scientific. Alleles were detected by cleavage of FAM or VIC reporter dye from the 5’ end of the oligonucleotide by Taq DNA polymerase. PCR reactions were carried out in a total volume of 25μL, including 0.63 μL of Probe, 12.5 μL of Taqman Universal Master Mix II (DNA polymerase, uracil-N glycosylase, dNTPs with dUTP, passive reference, and optimized buffer components), 6.87 μL of distilled water, and 50 ng of genomic DNA. The PCR reaction conditions were 95°C for 10 minutes (pre-degeneration), 35 cycles at 93°C for 40 seconds (denaturation), and 60°C for 1 minute (annealing/extension). Each PCR plate included positive and negative controls.
The three specific variants commonly found in the general population are rs1801133 (C677T), rs1801131 (A1298C), and rs2274976 (G1793A) [22]. The former two may decrease MTHFR enzyme activity with increased variant alleles [23]. All three sites code for nonsynonymous mutations in amino acid sequences.
Statistical analysis
We divided the polymorphism analysis into single-site and multi-site modes. For each SNP, genotype was entered as 0, 1, or 2 depending on the number of variant alleles. To provide a comprehensive view, the cumulative effects of the identified risk SNPs were evaluated by summing the mutant allele numbers (ranging from 0 to 6).
For disease progression research, the three sites were analyzed respectively to compare the difference in genotype frequencies between the HR and CON groups. The multi-site effects on cognitive ability and risk ratings were also analyzed. The χ2 test was used to analyze variant differentiation among each population. For correlation analysis of polymorphism and clinical features (cognitive ability and SIPS scores), unary linear regression analysis was applied. All SNP variables were entered simultaneously into the model, and the total allelic score was identified independently to predict schizophrenia risk.
RESULTS
Demographic data
There were no significant sex and age differences between the two groups (p=0.386 and p=0.678). The amounts of CON and HR groups were 76 (47 males and 29 females) and 87 (48 males and 39 females), respectively, while the average age were 24.8 years (24.9 for males and 24.7 for females) in CON and 23.5 years for HR (23.1 for males and 23.9 for females).
MTHFR polymorphism and the HR group
All three sites achieved Hardy-Weinberg equilibrium (>0.05), supporting data reliability. SNP rs1801133 and rs1801131 demonstrated in-linkage disequilibrium (R2=0.20, D’=1). The distribution of MTHFR rs1801133 genotype frequencies differed significantly between the HR and CON groups. TT genotype was related to increased schizophrenia risk in the HR group compared to CC and CT. Similarly, the HR group showed a significantly higher variant T allele frequency. There were no significant differences in genotype or allele frequencies at rs1801131 and rs2274976 sites between the two groups. After stratification by gender, C677T polymorphism tended to significantly increase among the females of HR group (Table 1).
Based on the multi-site mode, the HR group exhibited significantly higher variant allele frequencies for the MTHFR gene compared to CON, indicating that increased MTHFR polymorphism was associated with a higher risk of schizophrenia. After stratification by sex, both males and females in the HR group exhibited higher polymorphism compared to CON, with a statistically significant difference for males (Table 2).
MTHFR polymorphism and cognitive ability
Working memory and social cognition were used to describe cognitive ability in the study. Working memory is a limited cognitive system that stores temporary information for processing and plays a critical role in behavior-related instructing processes. Social cognition primarily focuses on management, storage, and application of information related to others and the social state.
We conducted a linear regression analysis between variant allele amounts and cognitive scores for both HR and CON subjects. In the HR group, the social cognition and working memory scores decreased significantly with an increase in the variant allele amount in MTHFR (y=-3.285x+43.07, F=7.086, p=0.010 in Figure 1 and y=-3.377x+46.97, F=6.260, p=0.015 in Figure 2). The results indicated that higher MTHFR polymorphism may have a noticeable adverse effect on cognitive ability in HR populations for schizophrenia. In the CON group, there was no significant variation trend between MTHFR polymorphism and cognitive ability (p>0.05). A significant correlation was observed in male HR subjects but not in the CON group (Figures 1 and 2).
MTHFR polymorphism and SIPS
SIPS is used to assess the symptoms in the prodromal stage of schizophrenia and to screen potential CHR individuals [24]. In this study, the final ratings were a sum of 12 item scores, including positive symptoms, negative symptoms, disintegration symptoms, and general symptoms. Each item was rated between 0 (definitely disagree) and 6 (definitely agree). In the CHR group, variant allele amounts and SIPS scores were used to conduct a linear regression analysis, which indicated that the SIPS score tended to increase with the variant allele amounts in total, male, and female groups (Figure 3). This suggested that increased MTHFR polymorphism may aggravate the risk of schizophrenia.
DISCUSSION
For most psychiatric diseases, pathogenesis typically results from a combination of congenital genetic factors and acquired environmental influences. Monoamines play significant roles in mental disorders, and numerous medications have been developed to regulate this neurotransmitter pathway [25]. Methylfolate is a critical substrate for the synthesis of these neurotransmitters [26] catalyzed by MTHFR for synthesis, promoting methyl group generation in folate metabolism. The resulting metabolites are involved in DNA methylation, homocysteine metabolism, and other vital transmethylation reactions [18,27,28]. Low-functioning genetic variants of MTHFR can become rate-limiting [29]. For instance, previous research indicated that individuals homozygous for the wild-type C allele of MTHFR maintained normal enzyme activity, while homozygous for the mutant T variant experienced a 75% decrease in enzyme activity, with significant limitations in DNA methylation and homocysteine metabolism [23].
Genetic factors influence the entire progression of psychiatric conditions, from high-risk stages to full-blown schizophrenia [30]. Certain psychotic symptoms indicate potential disease progression that may develop into a prodromal stage. In clinical practice, primary prevention before disease onset is increasingly critical. Studying high-risk psychosis can offer novel insights for early identification, improving outcomes for physicians and patients. Literature reviews indicate that high-risk individuals for schizophrenia exhibit significant alterations in various clinical features compared to healthy controls. Cognitive ability, quality of life, and executive control significantly decline in high-risk individuals [31]. Physiological and structural research has reported brain structure abnormalities and aberrant functional connectivity in high-risk subjects for schizophrenia [32], suggesting several psychopathological bases for this population.
However, at a genetic level, studies on high-risk individuals for schizophrenia are still lacking. We selected the MTHFR gene to study its effects on the clinical features of HR psychosis. Our findings suggested that certain genetic traits are altered in HR individuals for schizophrenia. Specifically, the MTHFR polymorphism rs1801133 was more prevalent in this population compared to healthy CON, supporting the notion that this MTHFR variant exacerbates schizophrenia risk. No significant differences were found for the other two sites. However, we found evidence of a cumulative effect involving MTHFR polymorphisms at C677T, A1298C, and G1798A, with more risk alleles across these sites in the HR population. While there is a lack of literature regarding genetic variants in individuals at a high risk for schizophrenia, our results were in agreement with the previous conclusions that MTHFR variant increases schizophrenia risk [33]. Our findings align with several existing studies about MTHFR polymorphism and schizophrenia, which suggested a higher risk of onset and worse cognitive ability [34,35[. Meanwhile, no significant correlation was also reported between this genetic factor and mental disorders in other literature [36]. Elaborate relationship is still underinvestigated.
In our previous study, multi-site MTHFR polymorphism was found to be more common in schizophrenic patients than in healthy controls [37]. These results suggested that MTHFR polymorphism may increase schizophrenia risk not only at disease onset but also in risk progression. The potential mechanism may involve alterations in folate and one-carbon metabolism processes, affecting homocysteine and methyl group conversion, which in turn influences DNA synthesis, gene expression, and epigenetic methylation modification. Two potential mechanisms may participate in the gene and disease risk association. Firstly, we interpret it as neurotransmitter pathway. It is widely acknowledged that level fluctuation of dopamine (DA), noradrenaline (NE), and serotonin (5-HT) is associated with mental disorders including schizophrenia and mood disorder [38]. The critical enzyme, tyrosine hydroxylase and tryptophan hydroxylase catalyze synthesis of DA, NE, and 5-HT. Both enzymes are activated by combination of tetrahydrobiopterin, while methylfolate is essential for the formation of this biological molecule [39]. As mentioned in previous, MTHFR activity decreases with mutant site like T allele of rs1801133, lowering the efficiency of one-carbon metabolism and inhibiting the synthesis of methylfolate and related downstream processes. Moreover, methyl groups would be supplied by one-carbon metabolism [22]. Altered MTHFR polymorphism and enzyme activity impacts on this reactivity and subsequently epigenetic modification of methylation, which transform the expression and regulation of susceptibility genes.
On stratification by sex, we found no significant differences in MTHFR single-site polymorphism within the HR group. However, in the multi-site analysis, the male subgroup exhibited higher MTHFR polymorphism in high-risk individuals compared to controls, while this was not observed in females. These findings are consistent with previous schizophrenia research, which reported sex differences, particularly in males [18]. The sex difference may be attributed to genetic factors, including sex chromosome-related gene expression regulation, or to sex-specific disease divergence in schizophrenia [40]. Sex chromosome inactivation is a critical potential mechanism to cause the gender differences. For female genome, there are twice X-linked genes on sex chromosome than males. One of the X chromosomes in female is ultimately inactivated (XCI) to keep the sex chromosome dosage balance between two genders. While this inactivation was skewed with 80% preference nonrandomly [41]. It was hypothesized that skewed XCI predisposes to regulate neuronal development, then influencing on the incidence and presentation of psychiatric diseases in females [42]. Overall, female psychotic individuals suffer more from sex chromosome related genes, while genes on autosome including MTHFR may take more effects on male ones. Biochemical factors might also play a role, as estrogen provides some protection for female schizophrenic patients by affecting neurodevelopment and social maturation, while testosterone may exacerbate the course of psychosis [43].
In addition to disease risk, we explored cognitive abilities across groups. A linear regression model was used to analyze the relationship between the number of risk alleles and cognitive scores in HR individuals and CON. Our findings suggested that an increase in the total number of MTHFR risk alleles contributes to the decline in working memory and social abilities in HR individuals for schizophrenia, supporting the notion that genetic variants have deleterious effects on cognitive ability. These results were consistent with previous findings, which indicated that high-risk groups exhibit a decline in several cognitive domains, including processing speed, attention, working memory, verbal learning, and social recognition [44]. Previous meta-analyses have also reported that high-risk subjects lag in all MCCB domains [9].
MTHFR polymorphism may influence folate metabolism and DNA synthesis, both crucial for neurodevelopment, thereby impacting neural development. Additionally, MTHFR-related changes in specific brain regions could contribute to cognitive and executive dysfunction [45]. Similar cortical and subcortical changes have been reported in high-risk subjects for schizophrenia [46], such as insular modification and amygdala hypoconnectivity [47]. Overall, these findings support a genetic and developmental hypothesis for the effects of cognition in HR individuals for schizophrenia.
For a more detailed investigation, we conducted a linear regression analysis on the relationship between risk allele amounts and SIPS scores in the CHR group. The results indicated that an increase in MTHFR polymorphism correlated with higher SIPS scores, suggesting that MTHFR variants have detrimental effects on the risk of psychosis. This trend was consistent across both male and female groups. The SIPS is a sensitive and specific tool for investigating high-risk psychosis, but studies examining its relationship with genetic factors are still scarce. Our findings, combined with our previous research, suggest that higher levels of MTHFR polymorphism in CHR individuals may lead to more severe prodromal symptoms of psychosis. This genetic feature in CHR individuals could potentially serve as a biomarker for primary prevention of schizophrenia, perhaps through the administration of supplements like methyl folate [48].
One-carbon pathway research offers a biological hypothesis for these mechanisms. More hypofunctional MTHFR variants lead to significantly decreased enzyme activity, resulting in low efficiency of the one-carbon pathway. This inefficiency disrupts folate and homocysteine metabolism and inhibits downstream methyl group supply and methylation modifications. Our previous study revealed alterations in both genomic and specific gene (SLC6A4) methylation in schizophrenic patients, supporting the involvement of epigenetic modifications in schizophrenia risk [19]. Genomic methylation analysis identified several genes with significant differential methylation positions and pathways correlated with schizophrenia, suggesting that they may be involved in the risk progression of the disorder.
Importantly, identifying novel biomarkers for basic research and clinical applications is valuable, given the need for objective criteria in recognizing and intervening in schizophrenia risk. Future studies may focus on methylation and related protein expression in CHR subjects for schizophrenia. This molecular study could offer insights into the mechanisms of disorder development and support preliminary screening in HR populations.
This study had several limitations. First, the sample size was insufficient, and, as with all genetic association studies, findings should be viewed as preliminary until replicated. Second, more hypofunctional variant sites in one-carbon metabolism should be investigated for schizophrenia risk, as each variant may affect the amino acid composition of associated proteins relevant to schizophrenia risk. Third, the influence of previous psychotropic medication should be considered since CHR subjects with prodromal symptoms may have been medicated, and such medication could affect methylation and related processes [49]. Moreover, potential selection bias and more compatible cross-sectional design should be explicitly acknowledged. Overall, subsequent studies on related methylation or protein levels are necessary to explore the distinct effects on the risk progression of schizophrenia.
This pilot study demonstrated that increased MTHFR polymorphism was associated with risk progression of schizophrenia. This association was more significant in males than in females. An increase in the hypofunctional MTHFR variants decreased the cognitive ability of both CHR and healthy subjects, indicating a greater risk in CHR subjects. The MTHFR polymorphism could promote the progression effects on HR psychosis before schizophrenia. Relevant genetic variants exhibit capacity of potential biomarker for clinical judgment of schizophrenia. Prospective work is critical on effector proteins and mechanism pathways to integrate the whole genetic theory.
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