Next Article in Journal
Imbalance and Falls in Patients with Parkinson’s Disease: Causes and Recent Developments in Training and Sensor-Based Assessment
Previous Article in Journal
Identifying the Qualities of Attention and the Attentional Style in Indoor Team Sports: A Gender Comparison
Previous Article in Special Issue
Rett Syndrome and the Role of MECP2: Signaling to Clinical Trials
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Brain Sciences
Volume 14
Issue 7
10.3390/brainsci14070624
brainsci-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Co-Occurring Methylenetetrahydrofolate Reductase (MTHFR) rs1801133 and rs1801131 Genotypes as Associative Genetic Modifiers of Clinical Severity in Rett Syndrome

by
Jatinder Singh
1,2,3,
Georgina Wilkins
1,2,3,
Ella Goodman-Vincent
1,2,3,
Samiya Chishti
1,2,3,
Ruben Bonilla Guerrero
4,
Leighton McFadden
1,2,3,
Zvi Zahavi
5 and
Paramala Santosh
1,2,3,*
1
Department of Child and Adolescent Psychiatry, Institute of Psychiatry, Psychology and Neuroscience, King’s College London, London SE5 8AF, UK
2
Centre for Interventional Paediatric Psychopharmacology and Rare Diseases (CIPPRD), South London and Maudsley NHS Foundation Trust, London SE5 8AZ, UK
3
Centre for Interventional Paediatric Psychopharmacology (CIPP) Rett Centre, Institute of Psychiatry, Psychology and Neuroscience, King’s College London, London SE5 8AF, UK
4
Genetica Consulting Services, Laguna Niguel, CA 92677, USA
5
Myogenes Limited, Borehamwood WD6 4PJ, UK
*
Author to whom correspondence should be addressed.
Brain Sci. 2024, 14(7), 624; https://doi.org/10.3390/brainsci14070624
Submission received: 17 May 2024 / Revised: 18 June 2024 / Accepted: 19 June 2024 / Published: 21 June 2024
(This article belongs to the Special Issue Molecular Mechanisms of Brain Development and Psychiatric Diseases)
Browse Figure
Review Reports Versions Notes

Abstract

:
Aim: Remethylation disorders such as 5,10-methylenetetrahydrofolate reductase (MTHFR) deficiency reduce the remethylation of homocysteine to methionine. The resulting hyperhomocysteinemia can lead to serious neurological consequences and multisystem toxicity. The role of MTHFR genotypes has not been investigated in patients with Rett Syndrome (RTT). In this study, we sought to assess the impact of co-occurring MTHFR genotypes on symptom profiles in RTT. Method: Using pharmacogenomic (PGx) testing, the MTHFR genetic polymorphisms rs1801133 (c.665C>T mutation) and rs1801131 (c.1286A>C mutation) were determined in 65 patients (18.7 years ± 12.1 [mean ± standard deviation]) with RTT as part of routine clinical care within the Centre for Interventional Paediatric Psychopharmacology (CIPP) Rett Centre, a National and Specialist Child and Adolescent Mental Health Service (CAMHS) in the UK. The clinical severity of patients was assessed using the RTT-anchored Clinical Global Impression Scale (RTT-CGI). Results: The clinical severity symptom distribution varied between the homozygous and heterozygous MTHFR rs1801133 and rs1801131 genotypes. Those with the homozygous genotype had a narrower spread of severity scores across several domains (language and communication, ambulation, hand-use and eye contact clinical domains). Patients with the homozygous genotype had statistically significantly greater CGI-Severity scores than individuals with a non-homozygous MTHFR genotype (Z = −2.44, p = 0.015). When comparing the ratings of moderately impaired (4), markedly impaired (5), severely impaired (6) and extremely impaired (7), individuals with the homozygous MTHFR genotype were more impaired than those with the non-homozygous MTHFR genotype (Z = −2.06, p = 0.039). There was no statistically significant difference in the number of prescribed anti-epileptic drugs between the genotypes. Conclusions: Our findings show that in those with a pathogenic RTT genetic variant, co-occurring homozygotic MTHFR rs1801133 and rs1801131 polymorphisms may act as associative genetic modifiers of clinical severity in a subset of patients. Profiling of rs1801133 and rs1801131 in RTT may therefore be useful, especially for high-risk patients who may be at the most risk from symptom deterioration.

1. Introduction

The level of unmet need for managing and treating mental health problems within the Rare Disease (RD) community is high. It is impossible to overstate the devastating consequences for those affected and their families, and the significant burden placed upon patients, families, health services, and society. Rett Syndrome (RTT) is an exemplar of a paediatric RD that encompasses both neuropsychiatric and physical problems. It is one of the most recognised models of abnormal synaptic plasticity [1]. Primarily caused by mutations in the protein methyl-CpG-binding protein 2 (MeCP2), RTT has a frequency of 5 to 10 cases per 100,000 females [2]. Where there is a pathogenic variant, the MECP2 gene is suggested to disrupt long gene expression within the brain [3], probably by altering topoisomerase IIβ activity [4]. Although the United Sates Food and Drug Administration (US, FDA) has recently approved Trofinetide in RTT therapeutics [5,6], typical treatment relies on the use of off-label medications [1], which can lead to ineffective doses being used, and may increase the risk of harm to patients and/or poor outcomes. Patients with RTT are at higher risk of adverse drug reactions (ADRs) due to the severity of the illness, co-occurring disorders, and the use of multiple medications. Indeed, in a prospective study of 18,820 adult patients in England, ADRs accounted for 6.5% of hospitalisations [7]. Polypharmacy and multimorbidity contribute to the higher incidence of ADRs [8]. In the UK, a small number of actionable pharmacogenes accounts for most prescription drugs, with 8/10 patients being exposed to at least one drug that was impacted by a variant in an actionable pharmacogene [9].
Methylenetetrahydrofolate reductase (MTHFR) deficiency involved in folate metabolism, specifically the synthesis of 5-methyl-tetrahydrofolate (5-MTHF), which is the biologically active form of folate required for the remethylation of homocysteine to methionine, has been associated with neuropsychiatric illness [10]. At present, there are about 247 single nucleotide polymorphisms (SNPs) of MTHFR documented in the Single Nucleotide Polymorphism Database (dbSNP) [11] and according to recent evidence, approximately 200 pathogenic MTHFR variants have been identified [12]. The genetic polymorphisms rs1801133 and rs1801131 are said to be important polymorphisms that have an impact on MTHFR activity [13]. While the selected genetic polymorphisms (rs1801133 and rs1801131) in this study are the only two with a clinical grade pharmacogenomics (PGx) level of evidence [14], these alleles are also related to disease risk [15,16,17,18,19,20,21]. Variants in MTHFR result in lower levels of 5-MTHF, and this increases homocysteine, thereby causing a dysregulated production of monoamines [22] and changes in DNA methylation [23]. In those with severe MTHFR deficiency, 109 mutations in 171 families have been described [24]. Severe MTHFR deficiency in children is characterised by hyperhomocysteinemia, hypotonia, feeding issues, failure to thrive, lethargy, apnoea, and microcephaly [24]. These findings are important in the context of RTT, especially given that others have indicated that there may also be progressive developmental delay, epilepsy, and impaired gait in patients with severe MTHFR deficiency [25]. These symptoms are thought to be caused in part due to reduced methylation within the brain [24]. Evidence has also suggested that the MTHFR c.665C>T variant increases the susceptibility to epilepsy [26]. An adolescent/adult-onset MTHFR deficiency with a focus on epilepsy manifesting as a neuropsychiatric illness has also been identified, and shares symptom overlap with RTT [27]. This study indicated that epilepsy occurs in about half of patients with adolescent/adult-onset MTHFR deficiency, and these patients have a highly variable response to antiepileptic drugs (AEDs) [27]. A few cases have also documented MTHFR deficiency, such as in a 14-year-old girl with progressive myoclonic epilepsy [28] and in AED-induced psychosis [29].
The UK Biobank study analysed PGx variant frequency in 487,409 subjects and showed that 99.5% of subjects had at least one actionable PGx variant [30] and the Pre-emptive Pharmacogenomic Testing for Preventing Adverse Drug Reactions (PREPARE) study further demonstrated a decrease in clinically relevant ADRs using a 12 gene PGx panel [31]. This evidence implies that there is also likely to be actionable PGx findings with potential treatment implications in the RTT patient population. However, neither the frequency nor the possibility of whether MTHFR rs1801133 and rs1801131 polymorphisms are genetic modifiers of clinical severity in RTT patients have been explored. Based on (I) the similarities of symptom profiles between MTHFR deficiency and RTT, (II) parallels in MECP2 and MTHFR methylation and (III) the finding that hyperhomocysteinemia over time causes irreversible neurological damage [27], it is useful to explore profiles of rs1801133 and rs1801131 polymorphisms in RTT patients. Progressive hyperhomocysteinemia would be especially important in RTT where there is already impaired methylation [3,4,32] and timely intervention would be critical to prevent further neurological decline. Preliminary reports have also suggested that hyperhomocysteinemia may itself be epileptogenic and lead to brain atrophy [33]. The overarching aim of this study was therefore to assess if MTHFR rs1801133 and rs1801131 polymorphisms are genetic modifiers of clinical severity in RTT. In this study, we thus (I) explored the frequency of MTHFR rs1801133 and rs1801131 polymorphisms in patients with RTT, (II) examined if homozygosity of these polymorphisms affects the symptom profile of RTT patients and (III) assessed if there is a relationship between these polymorphisms and AED use in patients.

2. Materials and Methods

2.1. Study Participants

Sixty-five (65) patients with RTT and a documented PGx test for MTHFR as part of their routine clinical care were evaluated in this study. Genetic diagnosis of RTT was confirmed using genetic reports or medical information, and where this information was unavailable, the caregiver was consulted. A clinical diagnosis based on the information provided by Neul et al. (2010) [34] was confirmed by either a Consultant Child and Adolescent Psychiatrist or an Associate Specialist specializing in RTT or from previous medical history/case notes. Where applicable, retrospective review of medical information determined the frequencies of co-occurring neuropsychiatric diagnoses and physical illness. The following descriptive data were also collected for each patient: age, gender, and race/ethnicity. The frequency of neuropsychiatric and physical illness for some of the patients in this study has been described previously [35]. For this study, we have provided the characteristics of neuropsychiatric and physical illness for all patients with an available PGx test for MTHFR.

2.2. Determination of MTHFR Variant Frequency

2.2.1. Sample Collection

After obtaining caregiver consent, PGx test kits (Myogenes, Borehamwood, UK), were posted to caregivers for sample collection. In some instances, sample collection was undertaken within the Centre for Interventional Paediatric Psychopharmacology (CIPP) Rett Centre. Samples were obtained from buccal swabs between April 2021 and January 2024. Testing was managed by Myogenes, who sent the samples for analysis to proprietary facilities such as Admera Health LLC and Innovative Gx Laboratories. Admera and Innovative Gx were two CLIA-approved and CAO-certified laboratories performing molecular testing. Both laboratories performed targeted PGx using next-generation sequencing.

2.2.2. PGx Test and MTHFR Reporting

PGx Test

After samples were analysed, a patient-specific PGx report was generated and sent to the referring physician (PS) for evaluation. Reports were discussed with the family with relevant guidance on medication prescribing. Where necessary, PS and JS received guidance from an expert physician in PGx (RBG) for a patient-specific interpretation of the results. The PGx reports contained information on (I) current medications and medications to be considered, (II) a comprehensive drug list and (III) genotype and phenotype results. In general, the comprehensive drug list included interactions for 67 genes and 294 medications that aligned with the Clinical Pharmacogenetics Implementation Consortium (CPIC), Food and Drug Administration (FDA), Association for Molecular Pathology (AMP), College of American Pathologists (CAP), American College of Medical Genetics (ACMG), and Pharmacogenomics Knowledgebase (PharmGKB) recommendations. The Innovative Gx Laboratories PGx panel genotyped 67 genes using next-generation sequencing (NGS) and qPCR with sensitivity and specificity values of 99.9% and 99.9%, respectively. The panel contained the specific genotype results of all the 67 genes. Two PGx reports were generated from Admera Health and included gene–drug interactions from a 62-gene panel.

MTHFR Reporting

Each PGx report provided information on two of the most common genetic polymorphisms of MTHFR rs1801133 (c.665C>T mutation) and rs1801131 (c.1286A>C mutation) with a clinical grade PGx level of evidence. The assignment of MTHFR rs1801133 and rs1801131 polymorphisms were conducted according to the predicted effect on MTHFR activity as described (mild, partial, or reduced activity) within each individual PGx report for the 65 patients. The designations were as follows:
1.
Homozygous designation (n = 17)
Individuals with rs1801133 (c.665 C>T) and rs1801131 (c.1286 A>C) homozygous genotypes that were predicted to have reduced MTHFR activity.
2.
Non-homozygous designation (n = 48)
Individuals with rs1801133 (c.665 C>T) and rs1801131 (c.1286 A>C) heterozygous genotypes that were predicted to have either mild or partial MTHFR activity (n = 41). Those predicted to have normal MTHFR activity were also assigned to this group (n = 7).

2.3. Assessment of Clinical Severity

To determine global clinical change, clinical severity was assessed using the RTT-anchored Clinical Global Impression Scale (RTT-CGI) and the following clinical domains for severity (CGI-S) were assessed: language/communication, ambulation, hand use, social [eye contact], autonomic, seizures and attentiveness [36].

2.4. Assessment of AEDs

The AEDs being prescribed to each patient at the time of the PGx test were collected. These findings were grouped by patients’ MTHFR genotype profiles. A Mann–Whitney U test was run to investigate whether the number of AEDs being taken at the time of the PGx test was statistically significant between the two MTHFR groups.

2.5. Data Extraction and Statistical Analyses

For each patient, data for MTHFR rs1801133 (c.665 C>T) and rs1801131 (c.1286 A>C) alleles (negative, heterozygote and homozygote) were extracted from individual PGx reports and transferred to an Excel spreadsheet. Based on CPIC and PharmGKB sources, the gene–drug information was also reviewed to gain further information on the relevance of the MTHFR phenotype on patient impact. Statistics and frequency histograms were obtained using Excel (Microsoft Excel, 2019). Due to the differing sample sizes between the homozygous population (n = 17) and the non-homozygous population (n = 48), a non-parametric test, the Mann–Whitney U test, was chosen as the analysis method (IBM SPSS Statistics 29.0.1.0).

2.6. Informed Consent and Ethics

Caregivers of all RTT patients in this study consented for their PGx testing to be carried out by Myogenes between April 2021 and January 2024 as part of routine clinical care. The PGx data to be collected and used for research and publication purposes received ethical approval as part of the Tailored Rett Intervention and Assessment Longitudinal (TRIAL) data warehouse by the London-Bromley Research Ethics Committee (REC reference: 15/LO/1772) on 19 October 2022 (substantial amendment number 7).

3. Results

3.1. Study Characteristics

Information for MTHFR rs1801133 and rs1801131 genotypes was available for 65 patients. Participant demographics are presented in Table 1A. All participants were female with a mean age (±SD) of 18.7 ± 12.1 years. In our sample population, 60 had RTT, 5 had atypical RTT, and 53 were white. Among the neuropsychiatric diagnoses, generalised anxiety disorder (45%) was the most common, followed by ASD (25%). Epilepsy (77%) was the most frequently occurring physical illness in the population, followed by gastrointestinal issues (68%) and breathing problems (52%) (Supplementary Information S1). The MTHFR rs1801133 and rs1801131 genotypes and their impact on MTHFR activity in the sample are presented in Table 1B. The normal, mild, partial, and reduced activity classifications for MTHFR were obtained from the PGx report for each patient.

3.2. Frequency of MTHFR rs1801133 and rs1801131 Genotypes in the Sample

The PGx panel reported on the two MTHFR genetic polymorphisms (rs1801133 and rs1801131) with a clinical grade PGx level of evidence. In our sample, 11% (n = 7/65), 63% (n = 41/65) and 26% (n = 17/65) were classified as normal, heterozygous, or homozygous, respectively. The frequency of the homozygotic genotype of rs1801133 (c.665C>T) was 35% (n = 6/17), while that of the homozygotic genotype of rs1801131 (c.1286A>C) was 65% (n = 11/17). The frequency of MTHFR genotypes stratified by age is shown in Supplementary Information S2.

3.3. Clinical Severity and MTHFR rs1801133 and rs1801131 Genotypes

Using the RTT-anchored CGI-S, we examined the symptom severity profile across the clinical domains for MTHFR rs1801133 and rs1801131 genotypes. This showed that the clinical severity profiles were different between the MTHFR homozygous and heterozygous genotypes (Table 2A). Varying patterns were noted across the clinical domains. A wider spectrum of severity was seen in the heterozygous individuals, whereas homozygous individuals were noted to have a narrower distribution for language and communication, ambulation, hand-use and eye contact clinical domains. The greatest difference in ambulation between the homozygous and heterozygous genotypes were noted at CGI-S:6 (60% homozygous vs. 31.8% heterozygous). Similarly, when looking at the clinical domain for hand use, a greater percentage of heterozygous individuals were concentrated at CGI-S:4 (45.4%), whereas homozygous individuals were more concentrated at CGI-S:6 (35.2% homozygous vs. 11.3% heterozygous). The clinical domain for autonomic symptoms showed a greater percentage of heterozygous individuals in CGI-S:4 (46.8% heterozygous vs. 17.6% homozygous). Interestingly, there was a greater percentage of homozygous individuals at CGI-S:6, which requires the presence of cyanosis (29.4% homozygous vs. 14.8% heterozygous). Furthermore, 21.2% of heterozygous individuals experienced no seizures compared to 5.8% of homozygous individuals. For the seizure clinical domain, the percentage of homozygous individuals was more concentrated at CGI-S:6 (35.2% homozygous) whereas the heterozygous individuals were more evenly distributed across all CGI-S ratings. Attentiveness profiles for homozygous and heterozygous individuals were similar, ranging between CGI-S:3 and CGI-S: 6 with the greatest difference noted at CGI-S: 5 (41.1% homozygous vs. 21.1% heterozygous). When examining overall RTT-anchored CGI-S ratings (Table 2B), the results indicated that individuals with a homozygous MTHFR genotype had significantly greater CGI-Severity scores than individuals with a non-homozygous MTHFR genotype (Z = −2.44, p = 0.015). When comparing the ratings of moderately impaired (4), markedly impaired (5), severely impaired (6) and extremely impaired (7), individuals with the homozygous MTHFR genotype were more impaired than those in the non-homozygous MTHFR group (Z = −2.06, p = 0.039).

3.4. AED Use and MTHFR Genotypes in RTT Patients

The use of the following AEDs was assessed between the homozygous and non-homozygous genotypes: Carbamazepine, Brivaracetam, Clobazam, Clonazepam, Lacosamide, Lamotrigine, Levetiracetam, Midazolam, Oxcarbazepine, Sodium Valproate, Topiramate and Zonisamide. An independent sample Mann–Whitney U test indicated no statistically significant difference between the number of AEDs prescribed for individuals in the homozygous MTHFR group when compared to individuals in the non-homozygous MTHFR group (Z = −0.56, p = 0.573) (Table 3). The frequency of the different AEDs used between the homozygous and the non-homozygous MTHFR group is presented in Supplementary Information S3.

3.5. RTT Diagnoses Categorised by MTHFR rs1801133 and rs1801131 Genotypes

Individuals with RTT were categorised according to the MTHFR rs1801133 and rs1801131 genotypes. There was no pattern between the mutation profile of RTT patients and the co-occurring MTHFR rs1801133 and rs1801131 polymorphisms tested (Supplementary Information S4).

4. Discussion

Our study, for the first time, has (I) identified and reported upon the frequency of co-occurring MTHFR rs1801133 and rs1801131 genotypes in patients with RTT and (II) demonstrated that those RTT patients with the homozygous MTHFR genotype have an overall worse symptom profile when compared to non-homozygous individuals as determined by the RTT-anchored CGI-S. This suggests that in a subset of RTT patients, co-occurring MTHFR rs1801133 and rs1801131 genotypes may act as associative genetic modifiers of clinical severity. However, this finding should be tempered because homocysteine and folate levels were not reported for our sample and the phenotypical correlates of individuals with MTHFR homozygosity are therefore of an associative level rather than a certainty of findings. Our follow-up work intends to measure homocysteine and folate levels from our patient group to explore the certainty of our findings and further examine how MTHFR rs1801133 and rs1801131 genotypes may act as associative genetic modifiers of clinical severity in RTT. Notwithstanding this limitation, this is the first study that has profiled MTHFR polymorphisms and its association with symptom severity in a paediatric neurodevelopmental disorder, in this case RTT, as part of routine clinical care in a National and Specialist CAMHS in the UK.
Individuals with the heterozygous or homozygous c.665C>T genotype have enzymatic activity 67–65% and 30–25% compared to the normal phenotype, respectively [12,37]. For those with c.1286A>C, the MTHFR activity is 83% and 61% for heterozygous and homozygous genotypes when compared to those with normal MTHFR activity [14]. The MTHFR c.665C>T and c.1286A>C genotypes vary across populations and depend on ethnicity [38]. In one study of 1405 subjects in the US, the highest c.665C>T variant frequency was found in Hispanics and Caucasians [39]. This aligns with the data from the 1000 Genomes projects that showed the highest frequency of c.665C>T in Hispanics (47%), Europeans (36%) and South Asians (12%) [39,40]. In comparison, the frequency of c.1286A>C is the highest in Southeast Asians (42%) when compared to Europeans (31%) [39,40]. Other evidence has shown that the homozygosity of the c.665C>T genotype was 18%, while that of the homozygous c.1286A>C genotype was 12.5% [41]. In our total sample, 63% of the genotypes were heterozygous while 26% were homozygous. Of those showing homozygosity, 35% of the genotypes were homozygous for c.665C>T and 65% were homozygous for c.1286A>C. Caution is needed when extrapolating MTHFR rs1801133 and rs1801131 genotype frequencies from studies undertaken in other populations using neurotypical subjects [38,39,40,41] to the observed MTHFR frequencies in our sample.
It has been suggested that some AEDs such as oxcarbazepine and topiramate might exacerbate MTHFR deficiency symptoms by worsening remethylation [42]. We, therefore, wanted to assess whether there is a higher frequency of AED use in the homozygous MTHFR group. Our data showed no difference in the number of AEDs used between the homozygous group compared to those in the non-homozygous MTHFR group.
Both MTHFR and MECP2 have a function in methylation. However, it is unknown how impaired remethylation of homocysteine to methionine caused by reduced 5-MTHF underpins the global methylation changes regulated by pathogenic MECP2. Although our current findings are at an associate level, i.e., patients with the homozygous MTHFR genotype may synergistically contribute to the phenotype of patients with RTT, it was more than two decades ago when it was first surmised that low levels of 5-MTHF in Rett patients reduce folate transport in the CNS [43]. It was shown that the levels of 5-MTHF can be normalised with improved clinical outcomes following oral folinic acid supplementation. Following that work, there is further evidence to suggest folate receptor autoimmunity exists in RTT patients [44]. Low 5-MTHF was also said to be associated with epilepsy [45]. In that study, improvements in hand stereotypies were also observed following 1 year after folinic acid supplementation [45]. However, another study that assessed 5-MTHF levels in the cerebrospinal fluid (CSF) of 76 females with RTT showed no clinically significant changes in spinal 5-MTHF levels [46]. In young infants with severe MTHFR deficiency, early betaine treatment was suggested to prevent developmental psychomotor decline [47]. However, there have also been a few cases of brain oedema in patients with MTHFR deficiency treated with betaine [24]. Some others have suggested that folinic acid does not improve the clinical course of MTHFR deficiency [25]. When looking through the lens of neuropsychiatric disorders, folate supplements can improve clinical outcomes. A systematic review that looked at the efficacy of folate supplements in 23 studies of psychiatric disorders demonstrated that levomefolic acid or 5-methylfolate could improve clinical outcomes for patients with some psychiatric disorders [48]. However, neither betaine nor folate seem to objectively improve clinical outcomes in RTT patients, although parent-reported measures do show improvement in some clinical domains such as sleep, breathing issues, hand stereotypies, ambulation, and communication in children less than 5 years old [49]. Similarly, although folinic acid supplementation raises 5-MTHF in RTT patients, there are no significant improvements in clinical outcomes [50,51].
Further work would be needed to determine whether the MTHFR remethylation pathway is disrupted in RTT patients. However, the impaired dysregulation of methylation markers involved in the MTHFR pathway in RTT may support this notion [52]. Pre-emptive PGx testing to stratify RTT population groups for MTHFR genotypes could help to identify the appropriate patient subsets. As previously indicated, individuals with homozygotic genotypes should have their plasma methionine and plasma total homocysteine levels assessed. It would be prudent to perform these investigations as early as possible to see if there is a dysfunction of the remethylation pathway. Evidence indicates that early initiation of betaine or folate supplementation can improve neurodevelopmental outcomes in remethylation disorders [47,53,54]. Indeed, data from the European Network and Registry for Homocystinurias and Methylation Defects (E-HOD) registry demonstrated that pre-symptomatic diagnosis was a predictive factor for better neurodevelopment outcomes in patients with MTHFR deficiency [55].
Previous evidence has suggested genotype–phenotype correlations in RTT and some RTT variants are thought to have a more severe phenotype than others [56]. Clustering of RTT patients according to co-occurring MTHFR genotypes identified from PGx testing in our study did not reveal patterns concerning the prevalence of a particular type of RTT mutation. In a previous study examining the treatment of folate and betaine in RTT, although there were some differences in RTT variants between the placebo and treatment arms, there was insufficient statistical power to determine the impact of these variants [49]. In our study, the numbers are too small to examine whether a particular pathogenic RTT variant in the presence of co-occurring MTHFR polymorphism would have more impact on clinical severity. A larger sample would be required for a definitive conclusion; however, given the rare nature of RTT, it may not be possible to obtain sufficient numbers. Even in cases where genotype–phenotype relationships in RTT have been suggested, the clinical presentation can still be highly variable [57,58] and recent expert consensus guidelines for the management of RTT have also indicated that genotype–phenotype correlations in RTT are inaccurate [59]. The diagnosis of RTT is based on clinical criteria that are independent of molecular profiles [34,59]. Therefore, understanding genotype–phenotype correlations in the presence of co-occurring MTHFR polymorphism in RTT may not add value to the evidence base. It is likely that methylation would be the most modified in those with RTT and co-occurring MTHFR deficiency and this combination could have the greatest impact on neurodevelopmental outcomes (Figure 1).

5. Conclusions

While the phenotypical correlates of subjects with MTHFR homozygosity remain to be established, our findings do point towards an association between MTHFR rs1801133 and rs1801131 homozygosity and symptom profiles in RTT. This could be relevant for the most vulnerable patients who are at higher risk. However, homocysteine and folate levels were not measured, and therefore the findings of our study are of an associative nature. It would be interesting to investigate how these genotypes affect the concentration of homocysteine or folate levels in our sample. Age-related effects of PGx are important. Studies on paediatric PGx-guided prescribing are scarce, and three drugs (codeine, lansoprazole, and omeprazole) are suggested to have age-specific paediatric guidance [60]. Despite this, others have shown that PGx-guided treatment would be helpful for mood disorders and gastritis/esophagitis in the paediatric population [61]. Other exemplars of paediatric PGx implementation also exist [62].
As we move forward, genome-wide association studies (GWASs) could help in further confirming PGx-predisposing loci, especially in the case of MTHFR deficiency. However, only about 10% of GWASs have evaluated drug responses [63]. If there are rare variations of specific pharmacogenes, a GWAS may not have sufficient power to detect an association relevant to PGx guidance that will be clinically actionable [64]. Polygenic risk scoring might also help predict an individual’s response to medication [62]; however, in RTT, the small sample size would probably limit the ability to obtain accurate polygenic risk scores [65]. An alternative strategy would be to use pharmacoepigenomics to show how epigenetic modifications, i.e., methylation, have a role in treatment responses such as in the effectiveness of antidepressants and/or antidepressant response [66]. This strategy has shown some promise as hypomethylation in LINE-1 is suggested to be associated with poor response to risperidone in psychosis [67].

Limitations

Rare genetic variants of MTHFR may be present in our sample. Nevertheless, these rare variants would not necessarily be detected by PGx testing, and therefore more complex associations between MTHFR remethylation impairments and RTT could be missed. We also did not directly measure remethylation impairments reflected by the methylation markers such as 5-MTHF, methionine, S-adenosylmethionine (SAM); S-adenosylhomocysteine (SAH), which are suggested to have an important role in methylation in RTT [52] or folate levels. Therefore, the phenotypical correlates of the subjects with MTHFR homozygosity in our sample should be treated with caution.
A recent review has indicated bias for including Europeans in PGx research [68]. In a scoping review of 23,701 articles that searched for the terms “precision medicine” or “pharmacogenetics”, less than 5% stated the ethnicity of the sample [69]. The lack of PGx studies in ethnically diverse populations underscores the notion that the association between MTHFR rs1801133 and rs1801131 homozygosity and symptom profiles in RTT in our sample would not necessarily be reflective of RTT patients in other geographical regions. Future studies will need to implement PGx testing in diverse ethnic groups not only to understand different variant frequencies within these groups and their clinical implications but also to address gaps and health inequalities in PGx research.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/brainsci14070624/s1. Supplementary Information S1: Frequency (%) of comorbid diagnoses in the sample (n=65); Supplementary Information S2: Frequency (count) of MTHFR rs1801133 and rs1801131 genotypes in RTT stratified by age (n=65); Supplementary Information S3: Frequency of Different AED use and MTHFR rs1801133 and rs1801131 genotypes in RTT; Supplementary Information S4: Rett Syndrome variants and MTHFR rs1801133 and rs1801131 genotypes.

Author Contributions

J.S. and P.S. developed the idea of the study. J.S. analysed the demographic and frequency data, wrote drafts, and finalised the manuscript. G.W. and E.G.-V. collected information regarding the RTT-anchored CGI and extracted the relevant clinical data for the analyses under the guidance of the senior author P.S., J.S., S.C. and P.S. assessed and interpreted the PGx reports for Rett patients as part of routine clinical care. L.M. helped in procuring the PGx reports and reviewed the database. G.W. obtained the statistics. J.S. and P.S. reviewed the manuscript for intellectual content. R.B.G. and Z.Z. reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

The Centre for Interventional Paediatric Psychopharmacology (CIPP) Rett Centre funded by Reverse Rett (RE16403) partly funded this study.

Institutional Review Board Statement

Ethical approval was granted on 19 October 2022 by London-Bromley research ethics committee (REC reference: 15/LO/1772) Substantial Amendment no. 7. All patients were part of the CIPP Rett Centre (South London and Maudsley NHS Foundation Trust). Pharmacogenomic testing was performed as part of the treatment package for routine clinical care. Caregivers were provided with detailed information of the study before consent was obtained. Publication consent was obtained from caregivers for the data in this study to be collected as part of the Development and Validation of the TRIAL (Tailored Rett Intervention and Assessment Longitudinal) Database for Rett Syndrome—Development of the Data Warehouse (REC reference: 15/LO/1772).

Informed Consent Statement

Informed consent was obtained for all study participants. All participants were registered in the CIPP Rett Centre and had PGx testing as part of their routine clinical care.

Data Availability Statement

Data can be obtained upon reasonable request from the corresponding author.

Acknowledgments

We thank Federico Fiori for assisting with the statistics.

Conflicts of Interest

P.S. was a previous Principal Investigator (PI) in the Sarizotan (Protocol Number Sarizotan/001/II/2015; ClinicalTrials.gov Identifier: NCT02790034) and the Anavex Life Sciences Corp. (Protocol Number: ANAVEX2-73-RS-002) clinical trial on Rett Syndrome (RTT). P.S. is currently the PI in the Anavex Life Sciences Corp. (Protocol Number: ANAVEX2-73-RS-003) clinical trial. P.S. is the co-inventor of the HealthTrackerTM and is the Chief Executive Officer and shareholder in HealthTrackerTM. S.C. was a previous sub-investigator in the Sarizotan (Protocol Number Sarizotan/001/II/2015; ClinicalTrials.gov Identifier: NCT02790034) and the Anavex Life Sciences Corp. (Protocol Number: ANAVEX2-73-RS-002). S.C. is currently a sub-investigator in the Anavex clinical trial in patients with RTT (Anavex Life Sciences Corp. [Protocol Number: ANAVEX2-73-RS-003]). J.S. was a previous Trial Research Methodologist in the Sarizotan Clinical Trial (Protocol Number Sarizotan/001/II/2015; ClinicalTrials.gov Identifier: NCT02790034) and a Research Manager of the Anavex clinical trial for RTT (Life Sciences Corp. [Protocol Number: ANAVEX2-73-RS-002/003]). J.S. is also an advisor for Reverse Rett. G.W., L.M. and E.G.V. have no competing interests. J.S., G.W., E.G.V., S.C., R.B.G., L.M. and P.S. have no association with Myogenes Limited, Admera Health or Innovative Gx Health through employment, consultancies, share ownership or grants or any other type of funding. Z.Z. is the medical director of Myogenes Ltd.

References

  1. Banerjee, A.; Miller, M.T.; Li, K.; Sur, M.; Kaufmann, W.E. Towards a better diagnosis and treatment of Rett syndrome: A model synaptic disorder. Brain 2019, 142, 239–248. [Google Scholar] [CrossRef] [PubMed]
  2. Petriti, U.; Dudman, D.C.; Scosyrev, E.; Lopez-Leon, S. Global prevalence of Rett syndrome: Systematic review and meta-analysis. Syst. Rev. 2023, 12, 5. [Google Scholar] [CrossRef] [PubMed]
  3. Gabel, H.W.; Kinde, B.; Stroud, H.; Gilbert, C.S.; Harmin, D.A.; Kastan, N.R.; Hemberg, M.; Ebert, D.H.; Greenberg, M.E. Disruption of DNA-methylation-dependent long gene repression in Rett syndrome. Nature 2015, 522, 89–93. [Google Scholar] [CrossRef] [PubMed]
  4. Nettles, S.A.; Ikeuchi, Y.; Lefton, K.B.; Abbasi, L.; Erickson, A.; Agwu, C.; Papouin, T.; Bonni, A.; Gabel, H.W. MeCP2 represses the activity of topoisomerase IIβ in long neuronal genes. Cell Rep. 2023, 42, 113538. [Google Scholar] [CrossRef] [PubMed]
  5. USFDA. Available online: https://www.fda.gov/drugs/news-events-human-drugs/fda-approves-first-treatment-rett-syndrome (accessed on 30 December 2023).
  6. Neul, J.L.; Percy, A.K.; Benke, T.A.; Berry-Kravis, E.M.; Glaze, D.G.; Marsh, E.D.; Lin, T.; Stankovic, S.; Bishop, K.M.; Youakim, J.M. Trofinetide for the treatment of Rett syndrome: A randomized phase 3 study. Nat. Med. 2023, 29, 1468–1475. [Google Scholar] [CrossRef] [PubMed]
  7. Pirmohamed, M.; James, S.; Meakin, S.; Green, C.; Scott, A.K.; Walley, T.J.; Farrar, K.; Park, B.K.; Breckenridge, A.M. Adverse drug reactions as cause of admission to hospital: Prospective analysis of 18 820 patients. BMJ 2004, 329, 15–19. [Google Scholar] [CrossRef] [PubMed]
  8. Osanlou, R.; Walker, L.; Hughes, D.A.; Burnside, G.; Pirmohamed, M. Adverse drug reactions, multimorbidity and polypharmacy: A prospective analysis of 1 month of medical admissions. BMJ Open 2022, 12, e055551. [Google Scholar] [CrossRef] [PubMed]
  9. Kimpton, J.E.; Carey, I.M.; Threapleton, C.J.D.; Robinson, A.; Harris, T.; Cook, D.G.; DeWilde, S.; Baker, E.H. Longitudinal exposure of English primary care patients to pharmacogenomic drugs: An analysis to inform design of pre-emptive pharmacogenomic testing. Br. J. Clin. Pharmacol. 2019, 85, 2734–2746. [Google Scholar] [CrossRef] [PubMed]
  10. Wan, L.; Li, Y.; Zhang, Z.; Sun, Z.; He, Y.; Li, R. Methylenetetrahydrofolate reductase and psychiatric diseases. Transl. Psychiatry 2018, 8, 242. [Google Scholar] [CrossRef]
  11. Mallhi, T.H.; Shahid, M.; Rehman, K.; Khan, Y.H.; Alanazi, A.S.; Alotaibi, N.H.; Akash, M.S.H.; Butt, M.H. Biochemical Association of MTHFR C677T Polymorphism with Myocardial Infarction in the Presence of Diabetes Mellitus as a Risk Factor. Metabolites 2023, 13, 251. [Google Scholar] [CrossRef]
  12. Barretta, F.; Uomo, F.; Fecarotta, S.; Albano, L.; Crisci, D.; Verde, A.; Fisco, M.G.; Gallo, G.; Stagna, D.D.; Pricolo, M.R.; et al. Contribution of Genetic Test to Early Diagnosis of Methylenetetrahydrofolate Reductase (MTHFR) Deficiency: The Experience of a Reference Center in Southern Italy. Genes 2023, 14, 980. [Google Scholar] [CrossRef]
  13. El-Khawaga, O.Y.; Al-Azzawy, M.F.; El-Dawa, A.N.; ElSaid, A.M.; Mustafa, W.; Saad, M. Association study between genetic polymorphisms in MTHFR and stroke susceptibility in Egyptian population: A case–control study. Sci. Rep. 2024, 14, 114. [Google Scholar] [CrossRef]
  14. van der Pol, K.H.; Nijenhuis, M.; Soree, B.; de Boer-Veger, N.J.; Buunk, A.M.; Guchelaar, H.J.; Risselada, A.; van Schaik, R.H.; Swen, J.J.; Touw, D.; et al. Dutch pharmacogenetics working group guideline for the gene-drug interaction of ABCG2, HLA-B and Allopurinol, and MTHFR, folic acid and methotrexate. Eur. J. Hum. Genet. 2024, 32, 155–162. [Google Scholar] [CrossRef] [PubMed]
  15. Chaturvedi, P.; Tulsyan, S.; Agarwal, G.; Lal, P.; Agrawal, S.; Mittal, R.D.; Mittal, B. Relationship of MTHFR and NQO1 Pharmacogenetics and Chemotherapy Clinical Outcomes in Breast Cancer Patients. Biochem. Genet. 2015, 53, 211–222. [Google Scholar] [CrossRef]
  16. D’Angelo, V.; Ramaglia, M.; Iannotta, A.; Addeo, R. Pharmacogenetics of methotrexate in pediatric hematological neoplasm treatment: Does it need a personalized regimen based on MTHFR polymorphisms? Expert Rev. Hematol. 2014, 7, 517–519. [Google Scholar] [CrossRef] [PubMed]
  17. Salazar, J.; Altés, A.; del Río, E.; Estella, J.; Rives, S.; Tasso, M.; Navajas, A.; Molina, J.; Villa, M.; Vivanco, J.L.; et al. Methotrexate consolidation treatment according to pharmacogenetics of MTHFR ameliorates event-free survival in childhood acute lymphoblastic leukaemia. Pharmacogenom. J. 2012, 12, 379–385. [Google Scholar] [CrossRef]
  18. Miranda-Vilela, A.L. Role of polymorphisms in factor V (FV Leiden), prothrombin, plasminogen activator inhibitor type-1 (PAI-1), methylenetetrahydrofolate reductase (MTHFR) and cystathionine β-synthase (CBS) genes as risk factors for thrombophilias. Mini Rev. Med. Chem. 2012, 12, 997–1006. [Google Scholar] [CrossRef] [PubMed]
  19. González-Mercado, M.G.; Rivas, F.; Gallegos-Arreola, M.P.; Morán-Moguel, M.C.; Salazar-Páramo, M.; González-López, L.; Gámez-Nava, J.I.; Munoz-Valle, J.F.; Medina-Coss y León, R.; González-Mercado, A.; et al. MTRR A66G, RFC1 G80A, and MTHFR C677T and A1298C Polymorphisms and Disease Activity in Mexicans with Rheumatoid Arthritis Treated with Methotrexate. Genet. Test. Mol. Biomark. 2017, 21, 698–704. [Google Scholar] [CrossRef]
  20. Lv, S.; Fan, H.; Li, J.; Yang, H.; Huang, J.; Shu, X.; Zhang, L.; Xu, Y.; Li, X.; Zuo, J.; et al. Genetic Polymorphisms of TYMS, MTHFR, ATIC, MTR, and MTRR Are Related to the Outcome of Methotrexate Therapy for Rheumatoid Arthritis in a Chinese Population. Front. Pharmacol. 2018, 9, 1390. [Google Scholar] [CrossRef]
  21. Nishimoto, E.; Ito, Y.; Sakakibara, T.; Nishikubo, T. Early treatment using betaine and methionine for a neonate with MTHFR deficiency. Pediatr. Int. 2019, 61, 1265–1266. [Google Scholar] [CrossRef]
  22. Bottiglieri, T.; Laundy, M.; Crellin, R.; Toone, B.K.; Carney, M.W.P.; Reynolds, E.H. Homocysteine, folate, methylation, and monoamine metabolism in depression. J. Neurol. Neurosurg. Psychiatry 2000, 69, 228–232. [Google Scholar] [CrossRef] [PubMed]
  23. Friso, S.; Choi, S.-W.; Girelli, D.; Mason, J.B.; Dolnikowski, G.G.; Bagley, P.J.; Olivieri, O.; Jacques, P.F.; Rosenberg, I.H.; Corrocher, R.; et al. A common mutation in the 5,10-methylenetetrahydrofolate reductase gene affects genomic DNA methylation through an interaction with folate status. Proc. Natl. Acad. Sci. USA 2002, 99, 5606–5611. [Google Scholar] [CrossRef]
  24. Froese, D.S.; Huemer, M.; Suormala, T.; Burda, P.; Coelho, D.; Guéant, J.L.; Landolt, M.A.; Kožich, V.; Fowler, B.; Baumgartner, M.R. Mutation Update and Review of Severe Methylenetetrahydro-folate Reductase Deficiency. Hum. Mutat. 2016, 37, 427–438. [Google Scholar] [CrossRef] [PubMed]
  25. Huemer, M.; Mulder-Bleile, R.; Burda, P.; Froese, D.S.; Suormala, T.; Zeev, B.B.; Chinnery, P.F.; Dionisi-Vici, C.; Dobbelaere, D.; Gökcay, G.; et al. Clinical pattern, mutations and in vitro residual activity in 33 patients with severe 5, 10 methylenetetrahydrofolate reductase (MTHFR) deficiency. J. Inherit. Metab. Dis. 2016, 39, 115–124. [Google Scholar] [CrossRef]
  26. Wu, Y.-L.; Yang, H.-Y.; Ding, X.-X.; Zhao, X.; Chen, J.; Bi, P.; Sun, Y.-H. Association between methylenetetrahydrofolate reductase C677T polymorphism and epilepsy susceptibility: A meta-analysis. Seizure 2014, 23, 411–416. [Google Scholar] [CrossRef]
  27. Gales, A.; Masingue, M.; Millecamps, S.; Giraudier, S.; Grosliere, L.; Adam, C.; Salim, C.; Navarro, V.; Nadjar, Y. Adolescence/adult onset MTHFR deficiency may manifest as isolated and treatable distinct neuro-psychiatric syndromes. Orphanet J. Rare Dis. 2018, 13, 29. [Google Scholar] [CrossRef]
  28. D’Aco, K.E.; Bearden, D.; Watkins, D.; Hyland, K.; Rosenblatt, D.S.; Ficicioglu, C. Severe 5,10-methylenetetrahydrofolate reductase deficiency and two MTHFR variants in an adolescent with progressive myoclonic epilepsy. Pediatr. Neurol. 2014, 51, 266–270. [Google Scholar] [CrossRef]
  29. Shimura, M.; Yamada, H.; Takahashi, H.; Yamada, N.; Go, S.; Yamanaka, G.; Kawashima, H. Antiepileptic drug-induced psychosis associated with MTHFRC677T: A case report. J. Med. Case Rep. 2019, 13, 250. [Google Scholar] [CrossRef] [PubMed]
  30. McInnes, G.; Lavertu, A.; Sangkuhl, K.; Klein, T.E.; Whirl-Carrillo, M.; Altman, R.B. Pharmacogenetics at Scale: An Analysis of the UK Biobank. Clin. Pharmacol. Ther. 2020, 109, 1528–1537. [Google Scholar] [CrossRef]
  31. Swen, J.J.; van der Wouden, C.H.; Manson, L.E.; Abdullah-Koolmees, H.; Blagec, K.; Blagus, T.; Böhringer, S.; Cambon-Thomsen, A.; Cecchin, E.; Cheung, K.-C.; et al. A 12-gene pharmacogenetic panel to prevent adverse drug reactions: An open-label, multicentre, controlled, cluster-randomised crossover implementation study. Lancet 2023, 401, 347–356. [Google Scholar] [CrossRef]
  32. Lavery, L.A.; Zoghbi, H.Y. The distinct methylation landscape of maturing neurons and its role in Rett syndrome pathogenesis. Curr. Opin. Neurobiol. 2019, 59, 180–188. [Google Scholar] [CrossRef] [PubMed]
  33. Gorgone, G.; Caccamo, D.; Pisani, L.R.; Currò, M.; Parisi, G.; Oteri, G.; Ientile, R.; Rossini, P.M.; Pisani, F. Hyperhomocysteinemia in patients with epilepsy: Does it play a role in the pathogenesis of brain atrophy? A preliminary report. Epilepsia 2009, 50, 33–36. [Google Scholar] [CrossRef] [PubMed]
  34. Neul, J.L.; Kaufmann, W.E.; Glaze, D.G.; Christodoulou, J.; Clarke, A.J.; Bahi-Buisson, N.; Leonard, H.; Bailey, M.E.S.; Schanen, N.C.; Zappella, M.; et al. Rett syndrome: Revised diagnostic criteria and nomenclature. Ann. Neurol. 2010, 68, 944–950. [Google Scholar] [CrossRef] [PubMed]
  35. Singh, J.; Ameenpur, S.; Ahmed, R.; Basheer, S.; Chishti, S.; Lawrence, R.; Fiori, F.; Santosh, P. An Observational Study of Heart Rate Variability Using Wearable Sensors Provides a Target for Therapeutic Monitoring of Autonomic Dysregulation in Patients with Rett Syndrome. Biomedicines 2022, 10, 1684. [Google Scholar] [CrossRef] [PubMed]
  36. Neul, J.L.; Glaze, D.G.; Percy, A.K.; Feyma, T.; Beisang, A.; Dinh, T.; Suter, B.; Anagnostou, E.; Snape, M.; Horrigan, J.; et al. Improving Treatment Trial Outcomes for Rett Syndrome: The Development of Rett-specific Anchors for the Clinical Global Impression Scale. J. Child Neurol. 2015, 30, 1743–1748. [Google Scholar] [CrossRef] [PubMed]
  37. Burda, P.; Suormala, T.; Heuberger, D.; Schäfer, A.; Fowler, B.; Froese, D.S.; Baumgartner, M.R. Functional characterization of missense mutations in severe methylenetetrahydrofolate reductase deficiency using a human expression system. J. Inherit. Metab. Dis. 2017, 40, 297–306. [Google Scholar] [CrossRef] [PubMed]
  38. Kumar, J.; Das, S.K.; Sharma, P.; Karthikeyan, G.; Ramakrishnan, L.; Sengupta, S. Homocysteine levels are associated with MTHFR A1298C polymorphism in Indian population. J. Hum. Genet. 2005, 50, 655–663. [Google Scholar] [CrossRef] [PubMed]
  39. Graydon, J.S.; Claudio, K.; Baker, S.; Kocherla, M.; Ferreira, M.; Roche-Lima, A.; Rodríguez-Maldonado, J.; Duconge, J.; Ruaño, G. Ethnogeographic prevalence and implications of the 677C>T and 1298A>C MTHFR polymorphisms in US primary care populations. Biomark. Med. 2019, 13, 649–661. [Google Scholar] [CrossRef] [PubMed]
  40. Consortium, G.P.; Auton, A.; Brooks, L.D.; Durbin, R.M.; Garrison, E.P.; Kang, H.M. 1000 Genomes Project Consortium; A global reference for human genetic variation. Nature 2015, 526, 68–74. [Google Scholar]
  41. Chango, A.; Boisson, F.; Barbé, F.; Quilliot, D.; Droesch, S.; Pfister, M.; Fillon-Emery, N.; Lambert, D.; Frémont, S.; Rosenblatt, D.S.; et al. The effect of 677C-->T and 1298A-->C mutations on plasma homocysteine and 5,10-methylenetetrahydrofolate reductase activity in healthy subjects. Br. J. Nutr. 2000, 83, 593–596. [Google Scholar] [CrossRef]
  42. Belcastro, V.; Striano, P.; Gorgone, G.; Costa, C.; Ciampa, C.; Caccamo, D.; Pisani, L.R.; Oteri, G.; Marciani, M.G.; Aguglia, U.; et al. Hyperhomocysteinemia in epileptic patients on new antiepileptic drugs. Epilepsia 2010, 51, 274–279. [Google Scholar] [CrossRef]
  43. Ramaekers, V.; Hansen, S.; Holm, J.; Opladen, T.; Senderek, J.; Häusler, M.; Heimann, G.; Fowler, B.; Maiwald, R.; Blau, N. Reduced folate transport to the CNS in female Rett patients. Neurology 2003, 61, 506–515. [Google Scholar] [CrossRef]
  44. Ramaekers, V.T.; Sequeira, J.M.; Artuch, R.; Blau, N.; Temudo, T.; Ormazabal, A.; Pineda, M.; Aracil, A.; Roelens, F.; Laccone, F.; et al. Folate receptor autoantibodies and spinal fluid 5-methyltetrahydrofolate deficiency in Rett syndrome. Neuropediatrics 2007, 38, 179–183. [Google Scholar] [CrossRef]
  45. Ormazabal, A.; Artuch, R.; Vilaseca, M.A.; Aracil, A.; Pineda, M. Cerebrospinal fluid concentrations of folate, biogenic amines and pterins in Rett syndrome: Treatment with folinic acid. Neuropediatrics 2005, 36, 380–385. [Google Scholar] [CrossRef]
  46. Neul, J.L.; Maricich, S.M.; Islam, M.; Barrish, J.; Smith, E.O.; Bottiglieri, T.; Hyland, K.; Humphreys, P.; Percy, A.; Glaze, D. Spinal fluid 5-methyltetrahydrofolate levels are normal in Rett syndrome. Neurology 2005, 64, 2151–2152. [Google Scholar] [CrossRef]
  47. Diekman, E.F.; de Koning, T.J.; Verhoeven-Duif, N.M.; Rovers, M.M.; van Hasselt, P.M. Survival and psychomotor development with early betaine treatment in patients with severe methylenetetrahydrofolate reductase deficiency. JAMA Neurol. 2014, 71, 188–194. [Google Scholar] [CrossRef]
  48. Lam, N.S.K.; Long, X.X.; Li, X.; Saad, M.; Lim, F.; Doery, J.C.; Griffin, R.C.; Galletly, C. The potential use of folate and its derivatives in treating psychiatric disorders: A systematic review. Biomed. Pharmacother. 2022, 146, 112541. [Google Scholar] [CrossRef]
  49. Glaze, D.G.; Percy, A.K.; Motil, K.J.; Lane, J.B.; Isaacs, J.S.; Schultz, R.J.; Barrish, J.O.; Neul, J.L.; O’Brien, W.E.; Smith, E.O. A study of the treatment of Rett syndrome with folate and betaine. J. Child Neurol. 2009, 24, 551–556. [Google Scholar] [CrossRef]
  50. Hagebeuk, E.E.O.; Duran, M.; Koelman, J.H.T.M.; Abeling, N.G.G.M.; Vyth, A.; Poll-The, B.-T. Folinic acid supplementation in Rett syndrome patients does not influence the course of the disease: A randomized study. J. Child Neurol. 2012, 27, 304–309. [Google Scholar] [CrossRef]
  51. Temudo, T.; Rios, M.; Prior, C.; Carrilho, I.; Santos, M.; Maciel, P.; Sequeiros, J.; Fonseca, M.; Monteiro, J.; Cabral, P.; et al. Evaluation of CSF neurotransmitters and folate in 25 patients with Rett disorder and effects of treatment. Brain Dev. 2009, 31, 46–51. [Google Scholar] [CrossRef]
  52. Hagebeuk, E.E.; Duran, M.; Abeling, N.G.; Vyth, A.; Poll-The, B.T. S-adenosylmethionine and S-adenosylhomocysteine in plasma and cerebrospinal fluid in Rett syndrome and the effect of folinic acid supplementation. J. Inherit. Metab. Dis. 2013, 36, 967–972. [Google Scholar] [CrossRef]
  53. Huemer, M.; Diodato, D.; Schwahn, B.; Schiff, M.; Bandeira, A.; Benoist, J.F.; Burlina, A.; Cerone, R.; Couce, M.L.; Garcia-Cazorla, A.; et al. Guidelines for diagnosis and management of the cobalamin-related remethylation disorders cblC, cblD, cblE, cblF, cblG, cblJ and MTHFR deficiency. J. Inherit. Metab. Dis. 2017, 40, 21–48. [Google Scholar] [CrossRef]
  54. Huemer, M.; Diodato, D.; Martinelli, D.; Olivieri, G.; Blom, H.; Gleich, F.; Kölker, S.; Kožich, V.; Morris, A.A.; Seifert, B.; et al. Phenotype, treatment practice and outcome in the cobalamin-dependent remethylation disorders and MTHFR deficiency: Data from the E-HOD registry. J. Inherit. Metab. Dis. 2019, 42, 333–352. [Google Scholar] [CrossRef]
  55. Yverneau, M.; Leroux, S.; Imbard, A.; Gleich, F.; Arion, A.; Moreau, C.; Nassogne, M.; Szymanowski, M.; Tardieu, M.; Touati, G.; et al. Influence of early identification and therapy on long-term outcomes in early-onset MTHFR deficiency. J. Inherit. Metab. Dis. 2022, 45, 848–861. [Google Scholar] [CrossRef]
  56. Cuddapah, V.A.; Pillai, R.B.; Shekar, K.V.; Lane, J.B.; Motil, K.J.; Skinner, S.A.; Tarquinio, D.C.; Glaze, D.G.; McGwin, G.; Kaufmann, W.E.; et al. Methyl-CpG-binding protein 2 (MECP2) mutation type is associated with disease severity in Rett syndrome. J. Med. Genet. 2014, 51, 152–158. [Google Scholar] [CrossRef]
  57. Ham, A.L.; Kumar, A.; Deeter, R.; Schanen, N.C. Does genotype predict phenotype in Rett syndrome? J. Child Neurol. 2005, 20, 768–778. [Google Scholar] [CrossRef]
  58. Halbach, N.S.; Smeets, E.E.; Braak, N.v.D.; van Roozendaal, K.E.; Blok, R.M.; Schrander-Stumpel, C.T.; Frijns, J.; Maaskant, M.A.; Curfs, L.M. Genotype-phenotype relationships as prognosticators in Rett syndrome should be handled with care in clinical practice. Am. J. Med. Genet. Part A 2011, 158A, 340–350. [Google Scholar] [CrossRef]
  59. Fu, C.; Armstrong, D.; Marsh, E.; Lieberman, D.; Motil, K.; Witt, R.; Standridge, S.; Nues, P.; Lane, J.; Dinkel, T.; et al. Consensus guidelines on managing Rett syndrome across the lifespan. BMJ Paediatr. Open 2020, 4, e000717. [Google Scholar] [CrossRef]
  60. Aka, I.; Bernal, C.J.; Carroll, R.; Maxwell-Horn, A.; Oshikoya, K.A.; Van Driest, S.L. Clinical Pharmacogenetics of Cytochrome P450-Associated Drugs in Children. J. Pers. Med. 2017, 7, 14. [Google Scholar] [CrossRef]
  61. Roberts, T.A.; Wagner, J.A.; Sandritter, T.; Black, B.T.; Gaedigk, A.; Stancil, S.L. Retrospective Review of Pharmacogenetic Testing at an Academic Children’s Hospital. Clin. Transl. Sci. 2020, 14, 412–421. [Google Scholar] [CrossRef]
  62. Bousman, C.A.; Al Maruf, A.; Marques, D.F.; Brown, L.C.; Müller, D.J. The emergence, implementation, and future growth of pharmacogenomics in psychiatry: A narrative review. Psychol. Med. 2023, 53, 7983–7993. [Google Scholar] [CrossRef] [PubMed]
  63. Pirmohamed, M. Pharmacogenomics: Current status and future perspectives. Nat. Rev. Genet. 2023, 24, 350–362. [Google Scholar] [CrossRef] [PubMed]
  64. Gamazon, E.R.; Skol, A.D.; Perera, M.A. The limits of genome-wide methods for pharmacogenomic testing. Pharmacogenetics Genom. 2012, 22, 261–272. [Google Scholar] [CrossRef]
  65. Johnson, D.; Wilke, M.A.; Lyle, S.M.; Kowalec, K.; Jorgensen, A.; Wright, G.E.; Drögemöller, B.I. A Systematic Review and Analysis of the Use of Polygenic Scores in Pharmacogenomics. Clin. Pharmacol. Ther. 2021, 111, 919–930. [Google Scholar] [CrossRef] [PubMed]
  66. Micale, V.; Di Bartolomeo, M.; Di Martino, S.; Stark, T.; Dell’Osso, B.; Drago, F.; D’Addario, C. Are the epigenetic changes predictive of therapeutic efficacy for psychiatric disorders? A translational approach towards novel drug targets. Pharmacol. Ther. 2023, 241, 108279. [Google Scholar] [CrossRef]
  67. Marques, D.F.; Ota, V.K.; Santoro, M.L.; Talarico, F.; Costa, G.O.; Spindola, L.M.; Cogo-Moreira, H.; Carvalho, C.M.; Xavier, G.; Cavalcante, D.A.; et al. LINE-1 hypomethylation is associated with poor risperidone response in a first episode of psychosis cohort. Epigenomics 2020, 12, 1041–1051. [Google Scholar] [CrossRef]
  68. McInnes, G.; Yee, S.W.; Pershad, Y.; Altman, R.B. Genomewide Association Studies in Pharmacogenomics. Clin. Pharmacol. Ther. 2021, 110, 637–648. [Google Scholar] [CrossRef]
  69. Popejoy, A.B. Diversity in Precision Medicine and Pharmacogenetics: Methodological and Conceptual Considerations for Broadening Participation. Pharmacogenomics Pers. Med. 2019, 12, 257–271. [Google Scholar] [CrossRef]
Brainsci 14 00624 g001
Figure 1. Neurodevelopmental impact and methylation pathways. Abbreviations: 5-THF (5-tetrahydrofolate); MECP2 (methyl-CpG-binding protein 2 [MeCP2]) gene; MTHFR (methylenetetrahydrofolate reductase); RTT (Rett Syndrome); SAH (S-adenosylhomocysteine); SAM (S-adenosylmethionine). Methylation is expected to be normal in neurotypical individuals (I) and the neurodevelopmental outcomes are as observed in the general population. In individuals with RTT, methylation is disrupted due to a pathogenic MECP2 variant and possibly due to impaired methylation markers and these impairments result in neurological dysfunction (II). In those with abnormal MTHFR, methylation is altered, and neurodevelopmental outcomes are also affected (III). Methylation is the most disrupted in those individuals with RTT and probably co-occurring MTHFR homozygosity (IV). This genotype may have the greatest impact on neurodevelopmental outcomes.
Figure 1. Neurodevelopmental impact and methylation pathways. Abbreviations: 5-THF (5-tetrahydrofolate); MECP2 (methyl-CpG-binding protein 2 [MeCP2]) gene; MTHFR (methylenetetrahydrofolate reductase); RTT (Rett Syndrome); SAH (S-adenosylhomocysteine); SAM (S-adenosylmethionine). Methylation is expected to be normal in neurotypical individuals (I) and the neurodevelopmental outcomes are as observed in the general population. In individuals with RTT, methylation is disrupted due to a pathogenic MECP2 variant and possibly due to impaired methylation markers and these impairments result in neurological dysfunction (II). In those with abnormal MTHFR, methylation is altered, and neurodevelopmental outcomes are also affected (III). Methylation is the most disrupted in those individuals with RTT and probably co-occurring MTHFR homozygosity (IV). This genotype may have the greatest impact on neurodevelopmental outcomes.
Brainsci 14 00624 g001
Table 1. Sample (n = 65) descriptives. (A) Subject characteristics. (B) MTHFR rs1801133 and rs1801131 genotypes and predicted impact on MTHFR activity in 65 patients with RTT.
Table 1. Sample (n = 65) descriptives. (A) Subject characteristics. (B) MTHFR rs1801133 and rs1801131 genotypes and predicted impact on MTHFR activity in 65 patients with RTT.
(A)
Patients with a PGx Test (n = 65)
Diagnoses
Rett Syndrome60
Atypical Rett Syndrome5
Ethnicity
White: English, Welsh, Scottish, Northern Irish or British53
Mixed/Multiple ethnic groups8
Black/African/Caribbean/Black British1
Asian/Asian British2
Other ethnic groups1
Age (years)
Mean (min. max)18.7 (2.5; 47.5)
Standard deviation±12.1
(B)
MTHFR GenotypesPhenotype (MTHFR Activity) *n
rs1801133
(c.665 C>T)		rs1801131
(c.1286 A>C)
AGGTMild10
AGTTPartial15
GAGTPartial1
GATTPartial2
GGGTPartial13
TT Yes (reduced)1
AATTYes (reduced)5
GGGGYes (reduced)11
CC Normal1
GGTTNormal6
* Predicted MTHFR activity (mild, partial, or reduced activity) as described in the individual patient pharmacogenomic reports for 65 patients. Notes: [I]. The normal designation was given for those that were predicted to have normal MTHFR activity (n = 7). [II]. The heterozygous designation was given for those predicted to have either mild or partial MTHFR activity. Individuals who had the slightly reduced MTHFR phenotype were also categorized as partial (n = 41). [III]. The non-homozygous (rs1801133 and rs1801131) descriptor was given to those predicted to have MTHFR activity as assigned for the normal and heterozygous groups (n = 48). [IV]. The homozygous (rs1801133 and rs1801131) descriptor was given for those with the most reduced MTHFR activity (n = 17).
Table 2. RTT Clinical Global Impression Scale Severity (CGI-S) ratings between non-homozygous and homozygous MTHFR rs1801133 and rs1801131 genotypes in RTT patients. (A) Number of patients (%) across different CGI-S clinical domains for MTHFR rs1801133 and rs1801131 genotypes. (B) Overall RTT-anchored CGI-S ratings.
Table 2. RTT Clinical Global Impression Scale Severity (CGI-S) ratings between non-homozygous and homozygous MTHFR rs1801133 and rs1801131 genotypes in RTT patients. (A) Number of patients (%) across different CGI-S clinical domains for MTHFR rs1801133 and rs1801131 genotypes. (B) Overall RTT-anchored CGI-S ratings.
(A)
CLINICAL
DOMAINS		CGI-S: 1CGI-S: 2CGI-S: 3CGI-S: 4CGI-S: 5CGI-S: 6CGI-S: 7
Language/
Communication		(CSS = 0)(CSS < 5)(CSS 5–10)(CSS 10–20)(CSS 20–25)(CSS 25–35)(CSS 35–40)
NormalAppropriate. May have unusual features such as perseveration/echolalia.
Reading disability/dyslexia		Phrase-sentences. May
have conversations or
echolalia		Words (<5). Babbles.
Makes choices. 25–50%		No words. Babbles. Makes choices ≤25%Vocalizations. Occasionally
screams. Makes no choices or only rarely
makes choices		No words. No vocalizations. Screams. No choices
Homozygous n = 160 (0%)0 (0%)0 (0%)5 (31.2%)5 (31.2%)6 (37.5%)0 (0%)
Heterozygous n = 430 (0%)0 (0%)2 (4.65%)11 (25.5%)13 (30.2%)15 (34.8%)2 (4.6%)
AmbulationNo impairmentNormal, may have slight
evidence of dystonia/ataxia/dyspraxia
upon careful examination		Walks, able to use stairs/run. May ride
tricycle or climb		Walks independently,
unable to use stairs or run		Walks with assistanceStands with support or independently.
May walk with
support. Sits independently
or with support		Cannot sit. Does not stand or
walk
Homozygous n = 150 (0%)0 (0%)0 (0%)2 (13.3%)2 (13.3%)9 (60%)2 (13.3%)
Heterozygous n = 440 (0%)1 (2.27%)6 (13.6%)7 (15.9%)12 (27.2%)14 (31.8%)4 (9.1%)
Hand UseCompletely normal, no impairmentNormal, may have slight
fine motor issues		Bilateral pincer
grasp. May use pen to write but has
some fine motor
issues like tremor		Reaches for objects, raking grasp or unilateral pincer.
May use utensils/cup		Reaches. No grasps.Rarely occasionally reaches out. No graspNone
Homozygous n = 170 (0%)0 (0%)0 (0%)5 (29.4%)5 (29.4%)6 (35.2%)1 (5.88%)
Heterozygous n = 441 (2.27%)0 (0%)2 (4.55%)20 (45.4%)15 (34.1%)5 (11.3%)1 (2.27%)
Social
(Eye Contact)		NormalOccasional eye gaze
avoidance		Appropriate eye
contact, >30 s		Eye contact
<20 s		Eye contact
<10 s		Eye contact,
inconsistent 5 s		No eye contact
Homozygous n = 140 (0%)0 (0%)1 (7.14%)7 (50%)6 (42.8%)0 (0%)0 (0%)
Heterozygous n = 390 (0%)5 (12.8%)9 (23.1%12 (30.7%)10 (25.6%)3 (7.69%)0 (0%)
AutonomicNoneMinimalNo or minimal
breathing abnormalities
(<5% of times
observed) and warm, pink extremities		Breathing dysrhythmia
<50%. No cyanosis.
Cool UE and LE pink		Breathing dysrhythmia
50%. No cyanosis.
Cool UE and LE pink		Breathing
dysrhythmia,
50–100%, maybe
with cyanosis.
Cold LE or UE,
may be blue		Breathing
dysrhythmia,
constantly with
cyanosis.
Cold UE and LE.
Mottled/blue
Homozygous n = 170 (0%)1 5.88%)4 (23.5%)3 (17.6%)4 (23.5%)5 (29.4%)0 (0%)
Heterozygous n = 470 (0%)0 (0%)6 (12.7%)22 (46.8%)12 (25.5%)7 (14.8%)0 (0%)
SeizuresNoneNone or controlledNone, with or
without meds		Monthly–
Weekly		WeeklyWeekly–DailyDaily
Homozygous n= 171 (5.8%)3 (17.6%)1 (5.88%)4 (23.5%)0 (0%)6 (35.2%)2 (11.7%)
Heterozygous n = 4710 (21.2%)8 (17.0%)5 (10.6%)9 (19.1%)5 (10.6%)6 (12.7%)4 (8.51%)
AttentivenessEntirely
normal		Occasional inattentionAttentive to conversation
and follows commands		50–100% of the time50% of the timeLess than 50% of the time0%
Homozygous n = 170 (0%)0 (0%)2 (11.7%)4 (23.5%)7 (41.1%)4 (23.5%)0 (0%)
Heterozygous n = 420 (0%)0 (0%)8 (19.0%)11 (26.1%)11 (26.1%)12 (28.5%)0 (0%)
(B)
RTT CGI-S
Clinical Domains		MTHFR rs1801133 and rs1801131 Genotypes
Non-Homozygous
(n = 47 *)		Homozygous
(n = 17)
Normal * (1)00
Borderline impaired (2)00
Mildly impaired (3)40
Moderately impaired (4)213
Markedly impaired (5)1610
Severely impaired (6)64
Extremely impaired (7)00
The difference in the number of patients in the clinical domains indicates that there was not sufficient clinical information available to complete the scoring. The colours of the clinical domains indicate increasing clinical severity. RTT-CGI-S provided in the work of Neul JL, Glaze DG, Percy AK, Feyma T and Beisang A. et al. entitled Improving Treatment Trial Outcomes for Rett Syndrome: The Development of Rett-specific Anchors for the Clinical Global Impression Scale. J Child Neurol. 2015 Nov;30(13):1743–8. reference [36]. * One individual did not have sufficient clinical information to complete the RTT CGI-S.
Table 3. Frequency of AED use and MTHFR rs1801133 and rs1801131 genotypes in RTT patients (n = 65).
Table 3. Frequency of AED use and MTHFR rs1801133 and rs1801131 genotypes in RTT patients (n = 65).
Number of AEDs *MTHFR rs1801133 and rs1801131 Genotypes
Homozygous (n = 17)Homozygous (%)Non-Homozygous (n = 48)Non-Homozygous (%)
None529%1531%
1741%2450%
2 424%715%
316%12%
400%00%
500%12%
Average number of AEDs used1.1 ± 0.9100%1.0 ± 0.9100%
* The AEDs prescribed to each patient at the time of the pharmacogenomics test.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Singh, J.; Wilkins, G.; Goodman-Vincent, E.; Chishti, S.; Bonilla Guerrero, R.; McFadden, L.; Zahavi, Z.; Santosh, P. Co-Occurring Methylenetetrahydrofolate Reductase (MTHFR) rs1801133 and rs1801131 Genotypes as Associative Genetic Modifiers of Clinical Severity in Rett Syndrome. Brain Sci. 2024, 14, 624. https://doi.org/10.3390/brainsci14070624

AMA Style

Singh J, Wilkins G, Goodman-Vincent E, Chishti S, Bonilla Guerrero R, McFadden L, Zahavi Z, Santosh P. Co-Occurring Methylenetetrahydrofolate Reductase (MTHFR) rs1801133 and rs1801131 Genotypes as Associative Genetic Modifiers of Clinical Severity in Rett Syndrome. Brain Sciences. 2024; 14(7):624. https://doi.org/10.3390/brainsci14070624

Chicago/Turabian Style

Singh, Jatinder, Georgina Wilkins, Ella Goodman-Vincent, Samiya Chishti, Ruben Bonilla Guerrero, Leighton McFadden, Zvi Zahavi, and Paramala Santosh. 2024. "Co-Occurring Methylenetetrahydrofolate Reductase (MTHFR) rs1801133 and rs1801131 Genotypes as Associative Genetic Modifiers of Clinical Severity in Rett Syndrome" Brain Sciences 14, no. 7: 624. https://doi.org/10.3390/brainsci14070624

APA Style

Singh, J., Wilkins, G., Goodman-Vincent, E., Chishti, S., Bonilla Guerrero, R., McFadden, L., Zahavi, Z., & Santosh, P. (2024). Co-Occurring Methylenetetrahydrofolate Reductase (MTHFR) rs1801133 and rs1801131 Genotypes as Associative Genetic Modifiers of Clinical Severity in Rett Syndrome. Brain Sciences, 14(7), 624. https://doi.org/10.3390/brainsci14070624

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop