Epigenetic alterations in folate transport genes in placental tissue from fetuses with neural tube defects and in leukocytes from subjects with hyperhomocysteinemia

Figures & data

Table 1. PCR primers forward (F), reverse (R) and sequencing primers (S) used for the FOLR1 gene Pyrosequencing methylation assays

CpG site No.	Nucleotide No.*	Primer sequence, 5′-3′	Ta (°C)	Size (bp)
1 & 2	116, 146	F: GGGTTAGGATTGAGTTTTTTAATGTTTG	59	93
5′CGI shore		R: biotin- AACCCACCTACTCATACAACTT
		S: TGAGTTTTTTAATGTTTGTATGAA
3–5	712, 716, 748	F:GTTTTTTTAAAGTGTGGGATTATAGAAATG	57.5	93
5′CGI shore		R: biotin-AAAAACACAACCCAAAATTTTAC
		S: GTGGGATTATAGAAATGAG
6−8	2508, 2518, 2524	F:GGTTAGGATGGTTTTGATTTTTTAGTT	52.9	263
CGI		R: biotin-ACCCCAAACTAAATACAATAACTTACTT
		S: AAAATGTTGGGATTATAGG
9–11	3457, 3466, 3477	F: GTTGGGATTTTTGAATTTGAGTTT	61	125
3′CGI shore		R: biotin-TCTTCCCACCATTACTCACAA
		S: TGTATTAAAATTATTTAGGTGGAT
12–14	3843, 3855, 3875	F:AGTGGGAGTTGTTTGTTAATTT	59	199
3′CGI shore		R: biotin-AACCCCACTCATAACTACAACATA
		S: ATTTTTATAAGGTTAGTAATTATAG

* Nucleotide number is relative to the ATG start site.

Table 2. PCR primers forward (F), reverse (R) and sequencing primers (S) used for the PCFT gene Pyrosequencing methylation assays

CpG site No.	Nucleotide No. *	Primer sequence, 5′-3′	Ta (°C)	Size (bp)
1–5 5′CGI shore	-4604, -4600,-4598 -4560, -4551	F: GGAAAAGAAAATTTGTATTTAGAGTGAGA R: biotin-ATCAAACTACTAACCTCAAATAATCC	57.5	170
		S: ATTTGTAAAAATAATAATATTTGGT
6–8	-2650, -2616, -2594	F:TTTGGGTTTTGGATTTTATAGTGTAG	55	177
5′CGI shore		R: biotin-ACTACCATATCCCCAACATCTAAAATAAT
		S: TTTTTTTTGAGATTGAGAGTT
9–31 CGI 1	-55, -50, -46, -44, -42, -37, -34, -30 -23, -14, -11, -9 11, 13, 22, 33, 35 39, 46, 53, 63, 70 79	F:GGTGGTTTTAGGTTATAGG R: biotin-ATTACTACAACCCCCCCTTTA S1: GTATATGGAGGGGAG S2: GTTGGTTTTAGGTAG	57.3	366
32–36 CGI 1	276, 287, 295, 304 318	F: GAGTTTGGTAGGTGGAGGGTT R: biotin-ATCAAACCCCTTTACTCTAATCCC	60	136
		S: AGGGTTTTGGTTTGG
38–44 CGI 2	687, 693, 716, 741 754, 757, 761, 779	F: TTTTTTATTGGATTTTTTATATGAA R: biotin-ACACTAAAACCTAAAACAACAAACC	55	147
		S1: TTTATTGGATTTTTTATATG
		S2: TGTTGGGAGTTTGGA
45–48 3′CGI shore	138, 1148, 1153 1162	F:TATGTAGTTTTTTGTTTTGGTGAGAT R: biotin-CCCAAAATACACAATAATCACCAC	60	177
		S: GGTGAGATTTTAAAGGAGTTA
49–50	2373, 2379	F:GTGTTGGGATTATAGGTATGAGT	60	170
3′CGI shore		R: biotin-AAAATTCCCTTCTACTTAATTATCAAAC
		S: GGGATTATAGGTATGAGTTAT
51–52	3926, 3931	F: AGATAGAGTAGGGTGAGTGTTAG	59	100
3′CGI shore		R: biotin-ACTATTCCCCCAACCATAAC
		S: GGTTTTTTATGTTTAGTGTTGT

* Nucleotide number is relative to the ATG start site.

Table 3. PCR primers forward (F), reverse (R) and sequencing primers (S) used for the RFC1gene Pyrosequencing methylation assays

CpG site No.	Nucleotide No. *	Primer sequence, 5′-3′	Ta (°C)	Size (bp)
1–3 5′CGI shore	-8067,-8042,-8025	F: GTTAGTGTTGGGAGGTTTGA R: biotin - TACAACTACCCCTCTCTCCA	59	181
		S: GTTTTTGTGATGGGTTAT
4–7 5′CGI shore	-7784, -7779, -7687,-7773	F: TGGAAATGTGGGAGGAAAAAT R: biotin-ACACCCCATATACAAAAAAAC	58	107
		S: ATTGTATATGTTGGGGTA
8–14 5′CGI shore	-6224, -6222,-6218 -6212, -6206,-6202 -6188	F: TGTGTGGTTGGGGAATTTT R: biotin-AAACCAATCCCTCACCTATCTC S: AGGGTAGTTTGGGTAGGTTTTT	57	131
15–17 5′CGI shore	-5456, -6030, -6010	F: GGAAGGGGGTGGGAGTTAT R: CCAAAACAACCTACTCCCTTTAC	57	119
		S: TTTGTGGGAAGGGGTTTA
18–23 CGI 1	-5205, -5199, -5193 -5184, -5182, -5171	F: TGGGAGAGTGGTTTAGGT R: biotin-AACCACCAATCCCCATCC	57	107
		S: GTTTTTTTTTTTGAGTGTGAT
24–30 CGI 1	-3713,-3704,-3693 -3688,-3664,-3649 -3629	F: GTGTAATTTTTTTTTGGAGTAGTTGTGG R: biotin-AACCCTAAAAAAATAACTTTCCTACTA	57	147
		S1: GTGTAATTTTTTTTTGGAGTAGTTGTGG
		S2: AGAGAGAGTTTGGATAG
31–33 3′CGI shore	-2989,-2974,-2953	F: TTTGGTTAGTTTTTTAGAGTAGGAGTTG R: biotin-ACCCCACTATCCACAATAAC	56	145
		S: GTAGGAGTTGGTTTAAAT
34–37 3′CGI shore	-2065,-2079,-2083 -2092	F: biotin-ATAGGTTTTGAGGAGGTATGG R: ACAACCAAAACCCTAAAAATTCTC	58	148
		S: AACCACTACCAAACC
38–42 5′CGI2 shore	-1082,-1067,-1049 -1037,-1032	F: GTGGTATTTGGAGATATGATTTTGATAAGT R: biotin-ACCCCAAACCACAAAACTTC	57.3	271
		S: ATTTTGATAAGTGGATAGGT
43–49 5′CGI2 shore	-971,-982,-988 -950,-939,-930 -918	F: biotin-TTGGGTAGAATTAGGATTGAAAG R: ACCCCAAACCACAAAACTT	57	159
		S1: AATAAAACAACAACAACAC
		S2: ACCCAACCTTAAAAACC
50–55	38,59,66,75,78,83	F: AGATTATTTTTTAAGGTGTTTTGATTTTAT	57.3	240
CGI 2		R: biotin-AAAATAATAAAACTCTCCCCTAACC
		S: GGTGGAGAAGTAGGTG

* Nucleotide number is relative to the ATG start site.

Figure 1. Methylated fraction in the FOLR1 gene in normal placenta (n = 4), leukocytes from subjects with low tHcy (c = 5–10 µmol/L, n = 24–25), and leukocytes from subjects with high tHcy (c = 20–113 µmol/L, n = 23–25). The error bars show ± 1 SD.

Figure 3. Methylated fraction in the RFC1 gene in normal placenta (n = 4), leukocytes from subjects with low tHcy (c = 5–10 µmol/L, n = 22–24), and leukocytes from subjects with high tHcy (c = 20–113 µmol/L, n = 21–24). The error bars show ± 1 SD. The upper panel shows CpG sites 1–24 and the lower panel shows CpG sites 25–55.

Table 4. mRNA expression of FOLR1, PCFT and RFC1 in full-term placental tissue and whole blood leukocytes

	FOLR1	PCFT	RFC1
Placental tissue	1.0 × 10^−5 ± 1.3 × 10^−5	1.4 × 10^−7 ± 8.5 × 10^−8	5.0 × 10^−6 ± 5.8 × 10^−5
WBC	8.4 × 10^−8 ± 1.2 × 10^−8	7.4 × 10^−6 ± 1.5 × 10^−5	1.3 × 10^−4 ± 2.9 × 10^−4
Ratio placenta/WBC	119	0.02	0.04

The relative expression was calculated with the ΔCt method (2^^{-∆ Ct}), using 18S as the reference gene.

Figure 2. Methylated fraction in the PCFT gene in normal placenta (n = 4), leukocytes from subjects with low tHcy (c = 5–10 µmol/L, n = 10–25), and leukocytes from subjects with high tHcy (c = 20–113 µmol/L, n = 11–25). The error bars show ± 1 SD. The upper panel shows CpG sites 1–30 and the lower panel shows CpG sites 31–52.

Table 5. Mean methylated fraction of CpG sites in the RFC1 gene in subjects with low or high plasma tHcy concentration

		Low tHcy			High tHcy
	CpG site*	Mean	SD	N	Mean	SD	N	p
5′shore	8	60.5	6.7	23	53.0	12.5	24	0.037
	9	72.5	6.3	23	64.2	13.2	24	0.022
	10	75.0	5.4	23	64.4	15.7	24	0.009
	13	73.0	5.5	23	64.5	13.6	24	0.017
	15	31.5	5.6	23	26.1	7.8	24	0.030
	16	56.0	7.0	23	47.8	8.9	24	0.002
	17	21.7	4.2	23	16.6	3.9	24	0.000
CGI 1	18	20.9	6.0	24	15.5	4.4	24	0.005
	21	10.5	3.5	24	7.5	1.5	24	0.024
	25	23.3	3.2	24	20.1	4.6	24	0.038
3′ shore	38	98.3	1.8	24	97.1	1.7	24	0.029
	43	74.4	5.9	23	78.5	2.6	22	0.010
	46	80.0	1.4	23	81.5	1.8	22	0.032
CGI 2	50	99.3	1.2	22	98.5	1.4	24	0.045
	52	98.5	1.4	22	97.2	1.7	24	0.007
	53	77.1	2.2	22	78.5	1.7	24	0.020
	55	97.3	2.2	22	95.9	1.9	24	0.025

* CpG sites included in the methylation assays, see Table 3. Only sites showing statistically significant differences are show in this table. P, post-hoc significance of difference between mean values in leukocytes from subjects with low or high tHcy, respectively.

Figure 4. Methylated fraction in the FOLR1, PCFT and RFC1 gene in placental tissue from healthy (n = 39–48) and NTD (n = 66–75) subjects. The error bars show 1 ± SD.

Table 6.RFC1 80G > A genotype prevalence and allele frequencies in subjects with low and high tHcy and normal and NTD subjects

		Leucocytes						Placenta
		Low tHcy			High tHcy			Normal			NTD
RFC1 80G > A		n	%		n	%		n	%		n	%
GG		6	(27)		8	(33)		8	(21)		22	(32)
GA		12	(54)		12	(50)		23	(61)		33	(49)
AA		4	(18)		4	(17)		7	(18)		13	(19)
Total		22			24			38			68
		χ^2 = 0.22			χ^2 = 0.02			χ^2 = 1.7			χ^2 = 0.01
p(G)		0.583			0.545			0.513			0.566
q(A)		0.417			0.454			0.487			0.434

Table 7. Methylated fraction of CpG sites in the RFC1 gene in blood leukocytes form subjects with low and high tHcy according to genotype

		Low tHcy				High tHcy
CpG site	Genotype	Mean	SD	N	p^1	Mean	SD	N	p^1	p^2
CpG 3	GG	89.3	0.5	6		89.9	0.6	8
	GA	89.3	0.7	12		89.8	0.8	12
	AA	89.0	0.8	4	0.656	90.0	0.8	4	0.823	0.006
CpG 6	GG	85.3	0.8	6		83.7	1.0	7
	GA	84.7	1.3	12		85.0	1.1	12
	AA	85.3	0.5	4	0.415	84.3	1.5	4	0.079	0.046
CpG 8	GG	56.8	8.6	6		50.3	11.4	8
	GA	61.0	6.2	12		56.3	8.5	12
	AA	61.0	1.0	3	0.448	48.5	23.2	4	0.434	0.029
CpG 9	GG	68.0	7.6	6		60.4	11.2	8
	GA	73.2	5.2	12		68.1	8.8	12
	AA	73.3	0.6	3	0.195	60.3	25.3	4	0.372	0.020
CpG 10	GG	72.2	7.6	6		58.8	16.1	8
	GA	75.6	4.5	12		69.5	9.4	12
	AA	76.0	3.6	3	0.432	60.5	27.4	4	0.291	0.007
CpG 11	GG	83.5	4.9	6		80.3	10.5	8
	GA	85.5	4.5	12		82.6	6.8	12
	AA	86.3	1.5	3	0.578	74.3	21.5	4	0.454	0.049
CpG 13	GG	69.5	6.4	6		59.5	14.5	8
	GA	73.5	5.3	12		68.8	7.9	12
	AA	74.0	0.0	3	0.307	61.3	23.1	4	0.293	0.014
CpG 15	GG	26.6	2.1	5		26.1	7.1	8
	GA	33.2	6.3	12		27.8	7.5	12
	AA	32.5	5.4	4	0.099	21.3	10.2	4	0.370	0.016
CpG 16	GG	52.2	4.4	5		46.9	7.8	8
	GA	56.3	7.6	12		50.4	7.0	12
	AA	59.5	8.7	4	0.332	41.8	14.4	4	0.233	0.001
CpG 17	GG	19.2	1.6	5		16.1	2.9	8
	GA	22.3	4.9	12		18.1	3.7	12
	AA	22.3	4.4	4	0.407	13.3	4.3	4	0.080	0.000
CpG 18	GG	16.2	2.6	6		15.1	4.1	8
	GA	22.3	6.4	12		17.4	2.8	12
	AA	23.5	7.2	4	0.089	10.8	5.7	4	0.021	0.000
CpG 19	GG	8.8	1.6	6		8.5	2.2	8
	GA	11.3	5.9	12		9.2	1.7	12
	AA	13.5	4.8	4	0.350	6.3	2.8	4	0.070	0.011
CpG 20	GG	8.0	1.4	6		7.6	2.0	8
	GA	10.1	5.5	12		8.5	1.9	12
	AA	12.3	4.4	4	0.370	5.3	2.8	4	0.042	0.012
CpG 21	GG	8.2	1.2	6		7.4	1.2	8
	GA	11.2	4.1	12		8.3	1.1	12
	AA	11.8	3.8	4	0.190	5.8	1.9	4	0.008	0.000
CpG 22	GG	3.3	0.5	6		3.3	1.6	8
	GA	5.6	3.1	12		4.3	0.9	12
	AA	6.5	2.4	4	0.132	2.8	1.3	4	0.052	0.012
CpG 24	GG	17.2	3.0	6		14.8	3.5	8
	GA	21.2	2.8	12		18.8	4.8	12
	AA	19.3	2.1	4	0.028	14.5	3.3	4	0.081	0.009
CpG 25	GG	21.3	2.9	6		18.1	4.8	8
	GA	24.3	3.4	12		21.6	4.1	12
	AA	22.8	1.7	4	0.186	19.8	5.1	4	0.265	0.026
CpG 31	GG	80.5	3.3	6		78.9	4.6	8
	GA	83.0	1.1	12		81.8	3.7	12
	AA	84.3	2.1	4	0.025	80.5	4.5	4	0.313	0.051
CpG 34	GG	91.0	1.2	4		90.0	1.3	6
	GA	92.8	0.9	10		92.2	1.4	11
	AA	93.3	1.0	4	0.010	93.3	1.9	4	0.006	0.232
CpG 38	GG	98.7	1.2	6		96.6	2.0	8
	GA	98.2	1.7	12		97.4	1.7	12
	AA	98.5	2.4	4	0.833	97.0	1.6	4	0.627	0.017
CpG 43	GG	73.4	8.3	5		77.8	2.9	6
	GA	76.7	4.0	12		78.6	2.7	12
	AA	71.3	6.4	4	0.228	79.3	1.7	4	0.703	0.003
CpG 46	GG	80.4	1.8	5		81.0	0.6	6
	GA	79.8	1.4	12		81.1	1.4	12
	AA	80.5	1.3	4	0.583	83.3	3.3	4	0.093	0.007
CpG 50	GG	99.7	0.8	6		98.4	1.4	8
	GA	99.2	1.3	12		98.7	1.6	12
	AA	99.0	1.4	4	0.646	98.0	1.4	4	0.730	0.044
CpG 51	GG	99.5	0.5	6		98.0	1.3	8
	GA	98.6	1.4	12		98.2	1.6	12
	AA	99.5	1.0	4	0.230	98.8	1.5	4	0.710	0.052
CpG 52	GG	98.0	1.7	6		97.0	1.5	8
	GA	98.4	1.4	12		97.4	1.8	12
	AA	99.3	1.0	4	0.402	96.8	1.7	4	0.756	0.006
CpG 53	GG	76.8	2.9	6		78.1	1.5	8
	GA	77.3	2.1	12		78.6	1.8	12
	AA	77.0	1.6	4	0.902	79.0	1.4	4	0.675	0.024
CpG 54	GG	76.2	2.6	6		77.9	1.1	8
	GA	38.8	1.9	12		39.2	1.6	12
	AA	3.3	1.0	4	0.000	2.5	0.6	4	0.000	0.417
CpG 55	GG	98.2	1.9	6		96.3	2.4	8
	GA	97.0	2.5	12		96.0	1.7	12
	AA	97.0	1.6	4	0.568	95.0	1.4	4	0.565	0.021

p¹ difference between mean methylated fraction in subjects with different RFC1 80G > A genotypes. p² difference between mean methylated fraction in subjects with high or low tHcy concentration, adjusted for RFC1 80G > A genotype. Please refer to the Statistics section for details of the multivariate model used.

Figure 5. Methylated fraction in the RFC1 gene according to RFC1 80G > A genotype in placentas, stratified by groups of healthy controls (n = 38) and NTD (n = 68). The error bars show ± SD.

Figure 6. Methylated fraction in the RFC1 gene in placentas of normal control births (n = 38) and NTD births (n = 68) stratified by RFC1 80G > A genotype.

Table 8. In-house design of mRNA expression assays for the PCFT and RFC1 genes

Gene		Sequence 5′-3′	Size (bp)
RFC1	Fw	CGTCAAGACCATCATCACTTTCA	100
	Rev	CAGGATCAGGAAGTACACGGAGTA
	Pr	CCGGTCCGCAAGCAGTTCCAGTT
PCFT	Fw	TTCACAGGATATGGGTTGCTTTT	120
	Rev	CACACAGGCCACAGCAGAAA
	Pr	CTGTCATCCGGGCTAAACTCTCCAAGCT

Fw, forward primer; Rw, reverse primer; Pr, probe