Methylenetetrahydrofolate Reductase Gene Variants Confer Potential Vulnerability to Autism Spectrum Disorder in a Saudi Community

Figures & data

Table 1 Epidemiologic and Clinical Characteristics in ASD Cases and Controls

Parameter	ASD Cases n = 112	Controls n = 104	Statistical Analysis
Age (years)
Age range	5‒22	6‒21
Mean	8.24	7.82	0.9 (−1.3‒0.5) (0.34)^a
SD	3.30	3.2
Gender
Male	96 (85.7)	88 (84.6)	1.1 (0.5–2.3) (0.82)^a
Female	16 (14.3)	16 (15.4)
IQ score:
Mean	57.1	62.1	5.0 (2.9‒7.1) (< 0.0001)^a
SD	8.2	7.5
CARS score
Range	31‒60	–
Mean	42.24		47.8 (40.7‒43.8) (< 0.0001)^a
SD	8.25
Mild/moderate (<37)^c	36 (32.1)		28.6 (22.9‒47.0) (< 0.0001)^b
Severe (≥37)^c	76 (67.9)
Excluded subjects:	n = 23 (parents refused to share)
	n = 17 (incomplete clinical data records)

Notes: ^aStudent t test (95% CI) (P value). ^bChi-square value [χ²] (95% CI) (P value). Bold numbers indicate statistically significant P values (P < 0.05). ^c Number of subjects, with percentages in parentheses.

Abbreviations: ASD, autism spectrum disorder; CARS, childhood autism rating scale; CI, confidence interval; IQ, intelligence quotient; SD, standard deviation.

Table 2 Genotype Distributions and Allele Frequencies of the 677C>T Rs1801133 and 1298A>C Rs1801131 SNPs in ASD Cases and Controls (Crude and Adjusted by Age)

Genetic Model	Interactive Genotype	ASD Cases^a n = 112	Controls n = 104	Logistic Regression (Adjusted Age)			Logistic Regression Crude Analysis
				OR (95% CI)	P Value^b	AIC^c	Or (95% CI)
MTHFR 677C≥T rs1801133:
Codominant	C/C	48 (42.9)	84 (80.8)	1			1
	C/T	48 (42.9)	20 (19.2)	0.23 (0.06–0.81)	0.0039	71.5	0.24 (0.07–0.84)
	T/T	16 (14.3)	0 (0.0)	0.0 (0.0-NA)			0.0 (0.0-NA)
Dominant	C/C	48 (42.9)	84 (80.8)	1			1
	C/T-T/T	64 (57.1)	20 (19.2)	0.17 (0.05–0.59)	0.0031	71.9	0.18 (0.05–0.61)
Recessive	C/C-C/T	96 (85.7)	104 (100)	1			1
	T/T	16 (14.3)	0 (0.0)	0.0 (0.0-NA)	0.019	75.2	0.0 (0.0-NA)
Overdominant	C/C-T/T	64 (57.1)	84 (80.8)	1			1
	C/T	48 (42.9)	20 (19.2)	0.30 (0.09–1.05)	0.050	76.8	0.32 (0.09–1.09)
Log-additive	—	—	—	0.19 (0.06–0.60)	0.0012	70.1	0.20 (0.06–0.61)
Allele:	C	144 (64)	188 (90)	1
	T	80 (36)	20 (10)	5.2 (3.1–8.9)	< 0.0001	NA	5.2 (3.1–8.9)
MTHFR 1298A≥C rs1801131:
Codominant	A/A	72 (64.3)	100 (96.2)	1			1
	A/C	12 (10.7)	4 (3.8)	0.24 (0.02–2.51)	0.0027	70.8	0.24 (0.02–2.50)
	C/C	28 (25.0)	0 (0.0)	0.00 (0.0-NA)			0.00 (0.0-NA)
Dominant	A/A	72 (64.3)	100 (96.2)	1			1
	A/C-C/C	40 (35.7)	4 (3.8)	0.07 (0.01–0.61)	0.0019	71	0.07 (0.01–0.61)
Recessive	A/A-A/C	84 (75.0)	104 (100.0)	1			1
	C/C	28 (25.0)	0 (0.0)	0.00 (0.00-NA)	0.0014	70.5	0.00 (0.00-NA)
Overdominant	A/A-C/C	100 (89.3)	100 (96.2)	1			1
	A/C	12 (10.7)	4 (3.8)	0.33 (0.03–3.44)	0.33	79.7	0.33 (0.03–3.43)
Log-additive	—	—	—	0.14 (0.02–0.83)	0.0008	69.4	0.14 (0.02–0.84)
Allele:	A	156 (70)	204 (98)
	C	68 (30)	4 (2)	22.2 (7.9–62.3)	< 0.0001	NA	22.2 (7.9–62.3)

Notes: ^aNumber of subjects, with percentages in parentheses. ^bP values were evaluated from logistic regression analysis after adjusting for age. Bold numbers indicate statistically significant P values (P < 0.05). ^cAIC values refer to the model with the less AIC value that corresponds to the minimal expected entropy. Underlined numbers represent the best models of inheritance with the less AIC value.

Abbreviations: ASD, autism spectrum disorder; MTHFR, methylenetetrahydrofolate reductase gene; AIC, Akaike information criterion; SNP, single nucleotide polymorphism; or, odds ratio; CI, confidence interval.

Table 3 Genotype Distributions and Allele Frequencies of the 677C>T Rs1801133 and 1298A>C Rs1801131 SNPs in ASD Patients According to CARS Scores (Adjusted by Age)

Genetic Model	Comparison	CARS Score <37 ^a n = 36	CARS Score ≥37 ^b n = 76	Logistic Regression
36 (32.1)	76 (67.9)			Or (95% CI)	P Value^c	AIC ^d
MTHFR 677C≥T rs1801133:
Codominant	C/C	8 (22.2)	40 (52.6)	1
	C/T	12 (33.3)	36 (47.4)	0.61 (0.08–4.59)	0.001	30.3
	T/T	16 (44.4)	0 (0.0)	0.00 (0.00-NA)
Dominant	C/C	4 (22.2)	40 (52.6)	1
	C/T-T/T	28 (77.8)	36 (47.4)	0.26 (0.04–1.58)	0.12	38.7
Recessive	C/C-C/T	20 (55.6)	76 (100)	1.0
	T/T	16 (44.4)	0 (0.0)	0.0 (0.0-NA)	0.004	32.1
Overdominant	C/C-T/T	24 (66.7)	40 (52.6)	1
	C/T	12 (33.3)	36 (47.4)	1.84 (0.35–9.71)	0.46	40.6
Log-additive	–	–	–	0.17 (0.04–0.76)	0.0064	33.7
Allele:	C	28 (39)	116 (76)	1
	T	44 (61)	36 (24)	5.1 (2.8–9.3)	< 0.0001
MTHFR 1298A≥C rs1801131:
Codominant	A/A	12 (33.3)	60 (79.0)	1
	A/C	0 (0.0)	12 (15.8)	NA (0.00-NA)	0.0014	30.0
	C/C	24 (66.7)	4 (5.3)	0.03 (0.00–0.39)
Dominant	A/A	12 (33.3)	60 (79.0)	1
	A/C-C/C	24 (66.7)	16 (21.1)	0.13 (0.02–0.78)	0.019	35.6
Recessive	A/A-A/C	12 (33.3)	72 (94.7)	1
	C/C	24 (66.7)	4 (5.3)	0.03 (0.00–0.32)	0.0005	29.0
Overdominant	A/A-C/C	36 (100)	64 (84.2)	1
	A/C	0 (0.0)	12 (15.8)	NA (0.00-NA)	0.12	38.6
Log-additive	–	–	–	0.21 (0.07–0.65)	0.0021	31.7
Allele:	A	24 (33)	132 (87)	1
	C	48 (67)	20 (13)	13.2 (6.7–26.0)	< 0.0001

Notes: ^aNumber of cases with CARS scores <37, with percentages in parentheses. ^bNumber of cases with CARS scores ≥37, with percentages in parentheses. ^cP values were evaluated from logistic regression analysis according to CARS scores after adjusting for ages. ^dAIC values refer to the model with the less AIC value that corresponds to the minimal expected entropy. Underlined numbers represent the best model of inheritance with the less AIC value. Bold numbers indicate statistically significant P value (P < 0.05).

Abbrevaitions: ASD, autism spectrum disorder; MTHFR, methylenetetrahydrofolate reductase gene; AIC, Akaike information criterion; CARS, childhood autism rating scale; SNP, single nucleotide polymorphism; or, odds ratio; CI, confidence interval; NA, not applicable.

Figure 1 Genotypic distributions of the 677C>T rs1801133 and 1298A>C rs1801131 SNPs in cases with CARS scores <37 and ≥37.

Table 4 Co-Segregation of Combined Genotypes of the MTHFR 677C>T Rs1801133 and MTHFR 1298A>C Rs1801131

Combined Genotypes		ASD Cases ^a n (%)	Controls ^a n (%)	Or (95% CI) (P Value)
MTHFR 677c>t	MTHFR 1298a>c
C/C	A/A	48 (42.9)	80 (76.9)	1 (reference)
C/C	A/C	0 (0.0)	4 (3.9)	0.2 (0.01–3.5) (0.26)
C/T	A/A	20 (17.9)	20 (19.2)	1.7 (0.8–3.4) (0.16)
C/T	A/C	12 (10.9)	0 (0.0)	41.5 (2.4–716.7) (0.01)
C/T	C/C	16 (14.5)	0 (0.0)	54.8 (3.2–933.8) (0.006)
T/T	A/A	4 (3.6)	0 (0.0)	14.9 (0.8–283.5) (0.07)
T/T	C/C	12 (10.9)	0 (0.0)	41.5 (2.4–716.7) (0.01)
Additive genotype comparison
C/C	A/A	48 (42.9)	80 (76.9)	1 (reference)
C/C	A/C+C/C	0 (0.0)	4 (3.9)	0.2 (0.01–3.5) (0.26)
C/T+T/T	A/A	24 (21.4)	20 (19.2)	2.0 (1.0–4.0) (0.049)
C/T+T/T	A/C+C/C	40 (35.7)	0 (0.0)	134.4 (8.1–2236.6) (0.0006)

Notes: Statistical analysis was based on Fisher’s exact t test. ^a Number of subjects, with percentages in parentheses. Bold numbers indicate statistically significant P values (P < 0.05).

Abbreviations: ASD, autism spectrum disorder; MTHFR, methylenetetrahydrofolate reductase gene; CI, confidence interval; or, odds ratio.

Table 5 Haplotype Association of the MTHFR 677C>T Rs1801133 and MTHFR 1298A>C Rs1801131 SNPs with Response to Study Groups (Adjusted by Age)

Haplotype	MTHFR 677c>t	MTHFR 1298a>c	Total	ASD Cases n = 112	Controls n = 104	Or (95% CI)	P Value
1	C	A	0.7206	0.5677	0.8846	1 (reference)	—
2	T	C	0.1187	0.2284	NA	0.00 (-infinity‒infinity)	1
3	T	A	0.1128	0.1287	0.0962	0.46 (0.13–1.63)	0.24
4	C	C	0.0479	0.0753	0.0192	39,011,699.2 (39,011,699.0–39,011,699.4)	< 0.0001
Haplotype association P value = 0.00057

Notes: Statistical analysis was based on logistic regression analysis. Bold numbers indicate statistically significant P values (P < 0.05).

Abbreviations: ASD, autism spectrum disorder; MTHFR, methylenetetrahydrofolate reductase gene; CI, confidence interval; or, odds ratio.

Table 6 Linkage Disequilibrium Analysis of MTHFR 677C>T Rs1801133 and MTHFR 1298A>C Rs1801131 SNPs

Linkage Disequilibrium Analysis	Frequency	Linkage Disequilibrium Measure
677C‒1298A (1) 677T‒1298C (2) 677T‒1298A (3) 677C‒1298C (4)	0.7206 0.1187 0.1128 0.0479	D` = 0.6256, r = 0.5097, r^2 = 0.260, P = 0.000

Notes: D`, linkage disequilibrium coefficient; “r”, correlation coefficient; “r²”, square of the correlation coefficient between two allelic variants. Bold numbers indicate statistically significant P values (P < 0.05).

Abbreviation: MTHFR, methylenetetrahydrofolate reductase gene.

Table 7 Correlation Between SNP Genotypes and Allelic Frequencies of MTHFR 677C>T and ASD Among Different Ethnic Populations

Population (Ethnicity)	Number of Cases (Controls)	Genotype Distribution of MTHFR 677C>T (%) Cases (Controls)			P Value	Variant Allele (%) Cases (Controls)	P Value	Reference
		C/C	C/T	T/T			T
Saudi Arabia (Gulf Arab)	112 (104)	43 (81)	43 (19)	14 (0)	0.0039	36 (10)	< 0.0001	Present study
American (mixed ethnic)	168 (5289)	21 (48)	56 (41)	23 (11)	< 0.0001	51 (32)	< 0.0001	[^Citation41]
American study (NA)	356 (205)	38 (45)	49 (44)	13 (11)	< 0.05	38 (33)	> 0.05	[^Citation42]
American (mixed ethnic)	138 (138)	71 (87)	25 (13)	4 (0)	< 0.0001	16 (7)	< 0.0001	[^Citation18]
Romania (central Europe)	39 (80)	54 (58)	36 (35)	10 (8)	0.85	31 (25)	0.60	[^Citation39]
Brazil (Latino)	151 (100)	40 (45)	45 (41)	15 (14)	0.72	38 (35)	0.77	[^Citation37]
Canada (Whites)	205 (384)	33 (46)	48 (43)	19 (11)	0.0016	43 (32)	0.0004	[^Citation19]
Han Chinese (East Asian)	186 (186)	43 (47)	41 (45)	16 (9)	0.03	37 (31)	0.02	[^Citation20]
Indian (Indian)	50 (50)	54 (84)	44 (16)	2 (0)	< 0.002	24 (8)	< 0.003	[^Citation43]
Korean (East Asian)	249 (423)	31 (33)	55 (48)	15 (19)	0.5303	42 (43)	> 0.05	[^Citation40]
Turkish (Turkish)	98 (70)	44.9 (53)	52 (47)	3 (0)	0.237	29 (24)	> 0.05	[^Citation38]
Egyptian (African Arab)	24 (30)	45.8 (67)	46 (27)	8 (7)	0.207	31 (20)	> 0.05	[^Citation22]
Egyptian (African Arab)	31 (39)	39 (90)	48 (10)	13 (0)	< 0.001	37 (5)	< 0.001	[^Citation44]

Note: Bold numbers indicate statistically significant P values (P < 0.05).

Abbreviations: MTHFR, methylenetetrahydrofolate reductase gene; NA, not available.

Table 8 Correlation Between SNP Genotypes and Allelic Frequencies of MTHFR 1298A>C and ASD Among Different Ethnic Populations

Population (Ethnicity)	Number of Cases (Controls)	Genotype Distribution of MTHFR 1298A>C (%) Cases (Controls)			P Value	Variant Allele (%) Cases (Controls)	P Value	Reference
		A/A	A/C	C/C			C
Saudi Arabia (Gulf Arab)	112 (104)	64 (96)	11 (4)	25 (0)	0.0012	30 (2)	< 0.0001	Present study
American (mixed ethnic)	168 (5289)	55 (44)^a	39 (47)	6 (9)	0.0005	25 (32)	> 0.05	[^Citation41]
American (mixed ethnic)	138 (138)	35 (48)	59 (32)	44 (58)	0.95	53 (54)	1.00	[^Citation18]
Canada (Whites)	205 (384)	53 (45)	40 (46)	7 (10)	0.124	27 (33)	0.059	[^Citation17]
Korean (East Asian)	236 (423)	62 (71)	32 (27)	6 (3)	0.021	22 (32)	< 0.05	[^Citation40]
Egypt (African Arab)	24 (30)	0 (40)	96 (53)	4 (6.6)	0.001	80 (20)	< 0.05	[^Citation20]
Egypt (African Arab)	31 (39)	23 (80)	42 (18)	36 (3)	< 0.001	57 (12)	< 0.001	[^Citation44]

Notes: ^aGenotype distribution is inconsistent with HWE. Bold numbers indicate statistically significant P values (P < 0.05).

Abbreviation: MTHFR, methylenetetrahydrofolate reductase gene.

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