Roles of 5,10-methylenetetrahydrofolate reductase C677T and A1298C polymorphisms in early- and late-onset obsessive-compulsive disorder.

BACKGROUND: The C677T and A1298C mutations of 5,10-methylenetetrahydrofolate reductase (MTHFR) have been linked with conditions such as depression, bipolar disorder, and schizophrenia, but there are not clear the relationship between MTHFR polymorphisms and obsessive-compulsive disorder (OCD). AIM: The current study was planned to investigate the link between the MTHFR polymorphisms and OCD in patients to reveal any potential correlations that may be used as a novel marker in diagnosis of people who are in high-risk group of developing OCD.
MATERIALS AND METHODS: Blood samples from 64 highly characterized symptomatic cases and 64 gender- and age-matched control participants were analyzed for MTHFR C677T and A1298C gene variants. The MTHFR gene polymorphisms were detected through real-time polymerase chain reaction, followed by melting curve analysis. The results were tested with analysis of variance test and the differences with P < 0.05 were reported as statistically significant.
RESULTS: A statistically significant difference in age, education level, and marital status was found in the comparison of all groups in sociodemographic findings (P = 0.004, P = 0.001, and P = 0.001, respectively). A statistically significant difference was found in the comparison of the tic story of early- and late-onset OCD patients (P = 0.002). There was no significant difference in the genotype frequencies and allele distributions of MTHFR polymorphisms between the patients and controls (P > 0.05).
CONCLUSION: The results suggest that MTHFR polymorphisms are unlikely to play a major role in the pathogenesis of OCD. Further studies are needed in biochemical data on folate metabolism to clarify the effect of the MTHFR polymorphisms in OCD pathophysiology.

INTRODUCTION

5,10-methylenetetrahydrofolate reductase (MTHFR) has key roles in purine and thymidylate biosynthesis through one-carbon metabolism (OCM) in DNA and amino acid methylations, RNA synthesis, and in neurotransmitter formation.[1] MTHFR helps channel OCM units between the DNA cycle and the methylation cycle through the OCM.[234] Decrease in MTHFR activity, which is involved in the homocysteine metabolism, affects homocysteine level, an indication of the role of MTHFR in folate metabolism. Mutations in MTHFR are known to cause hyperhomocysteinemia and homocystinuria causing various metabolic problems. The most common polymorphisms occurring in the MTHFR gene are C677T and A1298C. Since MTHFR affects methylation through S-adenosylmethionine, folate distribution, and homocysteine metabolism, genetic defects in MTHFR have a potentially negative and positive regulatory effect on disease risk.[567]

Psychiatric symptoms are reported to be related to Vitamin B12, folate, and homocysteine levels.[89101112] This fact shows the importance of Vitamin B12, folate, and homocysteine in carbon transfer (methylation), which is essential for the production of serotonin, catecholamines, and other monoamine neurotransmitters.[13] The link between folate deficiency and depressive disorders has been the topic of previous.[1415] Later, the focus moved on to the increased folate prevalence and Vitamin B12 deficiency in depressed patients.[1617] Observations on the antidepressant effects of folate may support the importance of folate in psychopathology.[1819] A few studies showed the effects of excessive OCM in anxiety disorders especially.[20212223] To the best of our knowledge, there are no studies on the relationship between MTHFR polymorphisms and obsessive-compulsive disorder (OCD). For this reason, we planned this study to investigate if the MTHFR polymorphisms in OCD patients are of etiopathogenetic significance and to show the effect of these polymorphisms at early- and late-onset of OCD.

MATERIALS AND METHODS

Patients

The study sample size was detected by power analysis using G-Power. Patients with a total of 64 OCD diagnoses according to the diagnostic and statistical manual of mental disorders V (DSM-V) diagnostic criteria according to clinical interview scales were divided into two groups according to the age of onset, early and late onset. Beginning age was taken as 15, and those who started after 15 years old were evaluated as late-onset OCD. As a healthy control, 64 patients with no OCD diagnosed and without psychotropic drugs or other medications were included in the study. Familial stories were assessed and those with psychiatric illnesses were recorded. Both healthy controls and OCD patients with schizophrenia, bipolar disorder, or high genetic transmission were not included in the study.

Ethics

This study was conducted in compliance with the good clinical practice and applicable regulatory procedure required by the Declaration of Helsinki. The study was approved by the Institutional Review Board (IRB) in May 2018. The IRB-approved informed consent forms were obtained from each participant before any study-related procedure.

Sociodemographic form and psychometrics tests

Sociodemographic findings of both groups were obtained by applying a sociodemographic data form. The Yale–Brown Obsessive-Compulsion Scale (Y-BOCS), Hamilton Depression Rating Scale (HAM-D), the Hamilton Anxiety Rating Scale (HAM-A), and the DSM-III-R personality disorders Structured Clinical Interview for DSM-II (SCID-II) were used to measure severity of illness in the OCD group. SCIs were administered for the DSM-IV Axis I Disorders (SCID-I).

DNA isolation

Genomic DNA was isolated from peripheral blood samples according to the manufacturer's instructions (Qiagen, Germany). DNA concentration was evaluated by absorbance methodology at 260 nm and DNA purity at 260/280 nm using a NanoDrop 1000 Spectrophotometer V3.7 (Thermo Scientific, USA).

Genotyping

SNPs were determined by real-time PCR using a panel of Light SNP assay from Way2 gene (assays based on Light Cycler HybProbe®). The Light Cycler HybProbe® in the LightSNP assay detects single-base mismatches, thus enabling the analysis of polymorphisms. The amplification process was followed by a melting curve analysis on Light Cycler 2.0 (Roche, Germany). Functional variants mapping in MTHFR gene C677T and A1298C polymorphisms were evaluated.

Data analysis

Amplification values of MTHFR C677T and A1298C genotypes were obtained at 640 nm. Mutant presence was detected at 54.5°C Tm for MTHFR C677T and the wild-type at 63.0°C. For A1298C, the mutant was detected at 58.5°C Tm and the wild-type at 49°C.

Statistical analysis

Data were analyzed by one-way analysis of variance and Chi-squared tests using SPSS-Windows 15 software package (IBM, Armonk, NY, USA).

RESULTS

Comparison of sociodemographical and clinical characteristics of the OCD and control groups is summarized in Table 1. Statistical differences in age and education level were present between the groups (P = 0.004 and P = 0.001, respectively). A statistically significant difference was also present in marital status between the groups (P = 0.001). There was no significant difference in gender distribution between the groups (P > 0.05). The difference in the tic story between the groups was also statistically significant (P = 0.002). There were no statistically significant differences in obsession type, compulsion type, triggered life story, starting story, HAM-D, HAM-A, Y-BOCS total, Y-BOCS Obsess (Y-BOCS obsessions), and Y-BOCS Comp (Y-BOCS compulsion) between the early- and late-onset OCD patients (P > 0.05) [Table 1].

Table 1

Demographical and clinical comparisons of groups

	EO-OCD group, n (%)		LO-OCD group, n (%)		Control group, n (%)		χ2	df	P
Gender
Female	25 (76.9)		27 (84.6)		38 (64.3)		3.04	2	0.217
Male	7 (23.1)		5 (15.4)		26 (35.7)
Marital status
Married	15 (23.1)		26 (73.1)		28 (75.0)		20.34	6	0.001*
Single	26 (69.2)		14 (23.1)		15 (21.4)
Divorced	1 (3.8)		0 (0.0)		1 (3.6)
Widow	1 (3.8)		1 (3.8)		0
Ticstory
Yes	14 (36.4)		6 (0.0)		0 (0.0)		9.38	1	0.002*
No	18 (63.6)		26 (100)		64 (100)
Triggered life event
Yes	16 (50)		24 (75)		-		4.69	1	0.07
No	16 (50)		8 (25)
Starting story
Sudden	6 (8.0)		9 (21.7)		-		1.81	1	0.237
Slow	26 (92.0)		23 (78.3)
Obsessions
Aggression	4 (8.2)		3 (6.1)				0.123	1	1.00
Contamination	16 (32.7)		20 (40.8)				2.34	1	0.19
Symmetry	10 (20.4)		10 (20.4)				0.014	1	1.00
Miscellaneous	8 (16.3)		2 (4.1)				4.22	1	0.04
Sexual	1 (2.0)		0				0.98	1	1.00
Religious	5 (10.2)		4 (8.2)				0.091	1	1.00
Somatic	2 (4.1)		1 (2.0)				0.313	1	0.51
Cleaning	16 (32.7)		12 (24.5)				0.980	1	0.24
Compulsions
Cleaning	16 (32.7)		19 (38.8)				1.380	1	0.34
Ritualistic	1 (2.0)		2 (4.1)				0.40	1	0.60
Counting	8 (16.3)		0				9.17	1	0.004
Ordering/arranging	10 (20.4)		10 (20.4)				0.014	1	1.00
Miscellaneous	8 (16.3)		2 (4.1)				4.22	1	0.07
Hoarding	6 (12.2)		2 (4.1)				2.20	1	0.24
	M	SD	M	SD	M	SD	F	df	P
Age	27.80	11.25	36.64	10.70	35.96	8.18	6.076	2	0.004*
Educational level (year)	11.12	3.4	9.52	4.84	9.6	13.76	7.733	2	0.001*
HAM-D	17.09	5.51	14.65	7.88	2.63	3.22	29.266	2	<0.0001
HAM-A	20.55	11.68	18.04	9.10	2.14	2.47	35.626	2	<0.0001
Y-BOCS total	24	6.25	22	6.78	0.0	-	6.420	2	0.956
Y-BOCS obsess	13.3	3.05	10.8	3.41	0.0	-	9.615	2	0.937
Y-BOCS comp	11.7	3.49	11.1	3.45	0.0	-	5.455	2	0.996

*Significant P<0.05. M – Mean average; SD – Standard deviation; Y-BOCS – Yale-Brown Obsessive-Compulsion Scale; Y-BOCS obsess – Y-BOCS obsessions; Y-BOCS comp – Y-BOCS compulsion; HAM-A – Hamilton Anxiety Rating Scale; HAM-D – Hamilton Depression Rating Scale; OCD – Obsessive-compulsive disorder; EO-OCD – Early-onset OCD; LO-OCD – Early-onset OCD

Based on the genotype analysis, the genotype distribution of MTHFR C677T gene polymorphism was 39% CC, 42% CT, and 19% TT in the OCD group, whereas it was 39% CC, 36% CT, and 25% TT in the control group. For MTHFR A1298C polymorphism, the genotype distribution was 30% AA, 54% AC, and 16% CC in the OCD group and 30% AA, 55% AC, and 15% CC in the control group. Underlying allele frequencies were calculated as 61% C, 39% T for C677T and 62% C, 38% T for A1298C variant in the early-onset OCD group. In the late-onset OCD group, the allele frequencies were 59% C, 41% T for C677T and 55% A, 45% A for A1298C variant. In the control group, the allele frequencies were 57% C, 43%T for C677T and 52% A, 48% A for A1298C variant. No significant difference was found with Chi-squared test between the early- and late-onset OCD group and the control group in terms of the genotype and allele distributions (P > 0.05) [Table 2]. There was also no significant difference between gender, marital status, tic story, obsessions, compulsions, Y-BOCS Total, Y-BOCS Obsess, Y-BOCS Comp, genotype, and allele distributions in the late- and early-onset OCD patients (P > 0.05) [Table 3].

Table 2

The genotype distributions and allele frequencies for methylenetetrahydrofolate reductase gene polymorphisms in groups

Groups	MTHFR C677T					P	MTHFRA1298C					P
	Genotype (n)			Allele frequency			Genotype (n)			Allele frequency
	CC	CT	TT	C	T		AA	AC	CC	A	C
EO-OCD	13	13	6	0.61	0.39	0.596	14	13	6	0.62	0.38	0.781
LO-OCD	12	14	6	0.59	0.41		10	14	7	0.55	0.45
Control	25	23	16	0.57	0.43		19	29	16	0.52	0.48

MTHFR – 5,10-methylenetetrahydrofolate reductase; OCD – Obsessive-compulsive disorder; EO-OCD – Early-onset OCD; LO-OCD – Early-onset OCD

Table 3

Comparison of 5,10-methylenetetrahydrofolate reductase gene polymorphisms with demographical and clinical features of groups

Groups	MTHFR C677T			P	MTHFR A1298C			P
	Genotype (n)				Genotype (n)
	CC	CT	TT		AA	AC	CC
Gender
Female	39	34	13	0.384	36	40	15	0.380
Male	18	16	8		12	16	9
Marital status
Married	29	27	13	0.384	22	32	16	0.937
Single	19	17	19		19	22	13
Divorced	1	1	0		1	1	0
Widow	1	1	0		0	2	0
Tic story
EO-OCD	26	15	8	0.197	15	21	4	0.942
LO-OCD	6	6	3		9	11	4
Obsessions
Aggression
EO-OCD	25	22	9	0.754	20	22	14	0.350
LO-OCD	10	9	5		8	11	5
Contamination
EO-OCD	13	8	6	0.210	8	12	6	0.164
LO-OCD	23	23	8		20	21	13
Symmetry
EO-OCD	20	16	7	0.210	15	19	9	0.583
LO-OCD	15	15	17		13	14	10
Miscellaneous
EO-OCD	25	23	8	0.208	19	24	13	0.198
LO-OCD	10	8	6		9	9	6
Sexual
EO-OCD	29	25	8	0.406	22	26	14	0.812
LO-OCD	6	6	6		8	8	5
Religious
EO-OCD	26	22	8	0.223	20	23	13	0.197
LO-OCD	11	11	8		8	10	6
Somatic
EO-OCD	29	25	8	0.406	21	28	13	0.418
LO-OCD	5	5	6		7	5	6
Compulsions
Cleaning
EO-OCD	12	10	7	0.290	19	20	13	0.164
LO-OCD	22	22	7		9	14	6
Ritualistic
EO-OCD	30	25	9	0.000	21	29	14	0.000
LO-OCD	5	7	5		7	5	5
Counting
EO-OCD	26	23	9	0.789	20	24	13	0.496
LO-OCD	9	9	5		8	10	5
Ordering/arranging
EO-OCD	22	18	7	0.508	15	22	10	0.410
LO-OCD	13	14	7		13	12	9
Hoarding
EO-OCD	25	24	8	0.192	19	25	13	0.188
LO-OCD	10	8	6		9	9	6
Miscellaneous
EO-OCD	28	23	7	0.160	20	25	13	0.946
LO-OCD	7	12	7		8	9	7
Y-BOCS total
EO-OCD	24	29	42	1.000	11	23	20	1.000
LO-OCD	26	20	5		10	22	19
Y-BOCS obsess
EO-OCD	26	30	42	1.000	11	23	20	1.000
LO-OCD	26	20	8		11	24	23
Y-BOCS comp
EO-OCD	24	30	42	1.000	11	23	20	1.000
LO-OCD	26	20	8		11	24	23

OCD – Obsessive-compulsive disorder; EO-OCD – Early-onset OCD; LO-OCD – Early-onset OCD; Y-BOCS – Yale–Brown Obsessive-Compulsion Scale; Y-BOCS obsess – Y-BOCS obsessions; Y-BOCS comp – Y-BOCS compulsion; MTHFR – 5,10-methylenetetrahydrofolate reductase

DISCUSSION

Psychiatric symptoms have been reported to be linked to Vitamin B12, folate, and homocysteine levels. Several studies have shown the effects of excessive carbon metabolism in anxiety disorders, especially OCD. Folate deficiency, high homocysteine levels, impaired methylation, and monoamine metabolism were reported in OCD patients by Atmaca et al.[21] Another study also reported high homocysteine levels and Vitamin B12 deficiency in some OCD patients.[24] One limitation of the current study is that serum levels of Vitamin B12, folate, and homocysteine were not measured in participants.

There are studies in the literature that question the association of OCD with folate, homocysteine, and Vitamin B12, but none with MTHFR gene polymorphisms. Considering the main role of MTHFR in brain function and neurodevelopment,[2526] common genetic variances are reported in psychiatric disorders through family and twin studies.[27282930] Therefore, this report investigated the potential link between OCD and the MTHFR gene polymorphisms.

Genetic modifications affecting the MTFHR level or function decreases 5-methyltetrahydrofolate level further resulting in increased homocysteine. Major depression was reported to be correlated with both improper folate metabolism and high homocysteine level, which may be due to the MTHFR polymorphisms.[313233] Moreover, MTHFR C677T and A1298C polymorphisms were linked to major depression and related treatment options.[333435] Since there are studies showing the relationship between MTHFR gene polymorphisms and major depression in the literature, severe depressive OCD patients having high HAM-D scores were excluded in this study. The average HAM-D score of the OCD participants in our study was 16 with only five participants having a HAM-D score higher than 27. According to these scores, the majority of our patients had mild depressive symptoms and five had moderate depressive symptoms. None of the participants were categorized to have major depressive disorder according to DSM-V diagnostic criteria. The fact that our participants had only mild and moderate depressive symptoms was another limitation of this study.

The link between MTFHR gene polymorphisms and the major psychiatric disorders, including bipolar disorder and schizophrenia, has been exhaustively studied. However, they were mostly inconclusive or reported mixed findings.[36373839404142] For example, in a study investigating the MTHFR C677T polymorphism on schizophrenia and affective disorder, it was concluded that the homozygote T allele does not play an important role in the pathogenesis of the diseases.[36]

There are many meta-analyses in the literature that reveal the relationship between psychiatric diseases and MTHFR gene polymorphisms. Two meta-analyses by Lewis et al.,[43] on 2427 participants and Muntjewerff et al.[44] on 4986 participants reported an elevated risk for schizophrenia with the TT genotype of MTHFR C677T polymorphism. The link between the MTHFR C677T polymorphism and several psychiatric disorders was also elucidated by two meta-analysis reports by Gilbody et al.[39] and Zintzaras.[45] The former report analyzed a total of 26 studies on schizophrenia (12 studies with over 6100 participants), bipolar disorder (4 studies with over 1600 participants), and unipolar depressive disorder (10 studies with over 11000 participants).[39] The latter report, on the other hand, covered two more articles on the top of what is covered by the former meta-analysis report.[40] Another meta-analysis report examined the links between the MTHFR C677T and A1298C polymorphisms with schizophrenia on over 9500 and 6100 participants, respectively.[46] Furthermore, the links between the MTHFR C677T and A1298C polymorphisms with bipolar disorder were examined on over 2200 and 700 participants, respectively. While the MTHFR C677T polymorphism was examined in all of these meta-analyses, the A1298C was only examined in some of them. One striking result pointed out by Peerbooms et al. in their meta-analysis with over 9600 patients and 19800 control participants was that although the TT genotype was represented in small percentage of the patients, it was significantly related with schizophrenia, bipolar disorder, and unipolar depressive disorder.[47] They also reported that the CC genotype of MTHFR A1298C polymorphism was also rare but significantly related with bipolar disorder not schizophrenia in their meta-analysis with over 3500 patients and 4400 control participants. In the current study, two polymorphisms of MTHFR showed approximately the same genotype profile in comparison of patient and control groups and early-/late-onset OCD groups. In comparison of the patient and control groups, the percentage of CT genotype in MTHFR C677T polymorphism was higher in OCD patients, but no statistical significance was detected in all comparisons.

Demographic differences in the participants may influence how MTHFR gene polymorphisms affect psychiatric disorders. Two studies revealed that demographic background and gender of participants might have close impact on the way MTHFR gene polymorphisms affect psychiatric disorders.[4548] In this study, there was no statistically significant difference between the demographic features and MTHFR gene polymorphisms of OCD cases.

CONCLUSION

This study was the first to investigate the relationship between early- and late-onset OCD and MTHFR gene polymorphisms, thus reflecting a section of the Turkish population. Our results lacked to show evidence that MTHFR gene polymorphisms play a role in the pathogenesis of OCD in Turkish participants. To clarify the effect of MTHFR C677T and A1298C polymorphisms in the OCD pathophysiology, the number of participants might be increased and biochemical data on folate metabolism might be investigated in the future studies.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.