Genotype-Based Epigenetic Differences in Monozygotic Twins Discordant for Positive Antithyroglobulin Autoantibodies

Abstract

Background:

Epigenetic factors associated with the development of autoimmune diseases are unclear. Monozygotic twin pairs discordant for positive antithyroglobulin autoantibodies (TgAb) are useful to examine the epigenetic factors because of their identical genetic background. This study aimed to clarify the discordant epigenetic differences affecting the development of TgAb.

Methods:

Subjects were selected from 257 Japanese monozygotic twins, recruited from the registry established by the Center for Twin Research at Osaka University. TgAb positive concordant (PC) pairs were 5.7% (four pairs) and 9.6% (18 pairs) of male and female pairs, respectively. TgAb discordant (DC) pairs were 11.4% (eight pairs) and 8.0% (15 pairs) of male and female pairs, respectively. TgAb negative concordant (NC) pairs were 78.6% (55 pairs) of male pairs and 74.3% (139 pairs) of female pairs. To perform stricter grouping, the cut-off value for positive TgAb was set to 50.0 IU/mL (TgAb negative: <28.0 IU/mL; TgAb positive: ≥50.0 IU/mL; TgAb borderline: ≥28.0 IU/mL and <50.0 IU/mL). Nineteen discordant (6 male and 13 female pairs) and 185 concordant pairs (48 male and 137 female pairs) for TgAb positivity were finally examined. DNA methylation levels of genomic DNA were evaluated using the Infinium HumanMethylation450 BeadChip Kit. Gene polymorphisms were also genotyped using the Omni5-4 BeadChip Kit to clarify genetic background specific for discordant twins.

Results:

No CpG sites were found with significant within-pair differences of methylation levels in TgAb DC pairs after correction for multiple comparisons. However, 155 polymorphisms specific for TgAb DC pairs were significantly different in genotype frequencies from those of concordant pairs, and none of them were located on the HLA region of chromosome 6. In TgAb DC pairs with some specific genotypes of these polymorphisms, four CpG sites were observed exhibiting significant within-pair differences in each DC pair, even after correction for multiple comparisons.

Conclusions:

This study found that the genetic background specific for TgAb DC twins who are susceptible to epigenetic changes are different from that specific for TgAb PC twins, and it clarified the genotype-based epigenetic differences in TgAb DC monozygotic twins.

Introduction

The predisposition to thyroid autoimmunity is believed to be affected by genetic and environmental factors. Many genome-wide association studies have found genetic factors susceptible to autoimmune thyroid diseases (AITD), including Graves' disease and Hashimoto's disease (1). The authors and other groups have reported that several gene loci such as CD40, CTLA4, PTPN22, FOXP3, and FCRL3 are associated with the development and prognosis of AITD (2,3). The development of antithyroid peroxidase autoantibodies (TPOAb) was also associated with some gene loci (4). Environmental factors such as stress, smoking, and excess iodine uptake can also be associated with the development and clinical presentations of AITD (5,6).

Monozygotic and dizygotic twins are very important resources to evaluate the relative contribution of genetic and environmental factors to phenotypes of a certain disease. Previous twin studies have reported that thyroid autoimmunity may be under strong gene influence and environmental factors (7,8). Model-fitting analysis showed that 79% of the liability of Graves' disease development is attributable to genetic factors and 21% to individual-specific environmental factors (9). It is very difficult to identify environmental factors affecting the predisposition to thyroid autoimmunity in the general population. However, it is easy to identify the environmental factors when examined in monozygotic twins discordant for positive thyroid autoantibodies who are susceptible to environmental factors. This study focused on epigenetic changes of DNA in order to estimate the impact of environmental effects on the predisposition to thyroid autoimmunity.

Modifications to genomic DNA, such as DNA methylation and histone modification, are typical epigenetic factors, and they impact cellular behavior by regulating chromatin status and gene expression (10). One of the most important epigenetic modifications is methylation of genomic DNA, which is the covalent addition of a methyl group to the cytosine in C-phosphate-G (CpG) dinucleotides. The CpG sites present in the regions containing high numbers of CpG dinucleotides (CpG islands) are generally unmethylated, whereas those in most other genomic regions are methylated. CpG islands overlap with the promoter regions of 60–70% of genes and are generally protected from methylation, allowing for the expression of downstream genes that are further regulated by histone modification (11). It was supposed that aberrant DNA methylation of CpG sites may be one of the most important epigenetic changes affecting the developmental process of autoimmunity, leading to regulation of gene expression. Discordant twins for type 1 diabetes have been already analyzed, and the results suggest that some abnormalities in DNA methylation patterns may be involved in the pathogenesis of diabetes (12). With regard to thyroid autoimmunity, the genetic–epigenetic interaction involving a noncoding polymorphism in the THSR gene have been reported using cultured thyroid cells (13). However, the epigenetic differences, including DNA methylation, in discordant phenotypes of monozygotic twins with AITD have not been investigated.

This study investigated levels of antithyroglobulin autoantibodies (TgAb), the most sensitive tests to detect thyroid autoimmunity, and the methylation levels of CpG sites in monozygotic (identical) twins to clarify the association between autoantibody production and methylation patterns of genomic DNA. Moreover, twins were also genotyped to find the genomic backgrounds specific for the discordant pair of TgAb positivity to look for individual differences in the sensitivity to epigenetic changes related to TgAb development.

Subjects and Methods

Subjects

A total of 257 Japanese monozygotic twins (70 male and 187 female pairs) were recruited from the registry established by the Center for Twin Research at Osaka University (14). Written informed consent was obtained from all the twins, and the Ethics Committee of Osaka University approved the study protocol (no. 506). Blood samples were taken at 9:00am after a 12 hour fast. A clinical examination was performed, and the twins filled in health-related questionnaires, including questions regarding thyroid disease. The twins were examined on the same day. Genomic DNA was isolated from peripheral blood mononuclear cells using a commercial kit (QIAamp DNA Mini Kit; QIAGEN, Hilden, Germany). Zygosity of subjects was confirmed by perfect matching of 15 short tandem repeat (STR) loci using the PowerPlex^® 16 System (Promega, Madison, WI).

TgAbs

TgAbs were examined with commercial electrochemiluminescence immunoassay (ECLIA) kits (ECLusys anti-Tg; Roche Diagnostics, Tokyo, Japan). The reference value for TgAb is <28.0 IU/mL

Methylation levels of CpG islands

Analysis of methylation levels was performed using the Infinium HumanMethylation450 BeadChip Kit (Illumina, San Diego, CA) according to the manufacturer's standard protocol, which interrogated 485,577 highly informative CpG sites at a single-nucleotide resolution for each sample. The experiment was performed with 0.5 μg of high-quality genomic DNA. On the chip, there were two bead types for each CpG site per locus. The raw data were analyzed by Genome Studio software (Illumina), and the fluorescence intensity ratios between the two bead types were calculated. A ratio value of 0 was equal to nonmethylation of the locus; a ratio of 1 was equal to total methylation. These raw data were corrected to normalize the differences in detection ranges between the two probes of the Infinium Assay using a peak-based correction method (15). Normalized data were filtered to exclude invalid probes such as null probes and probes with low reliability. R Statistical Software v3.1.2 (The R Foundation, Vienna, Austria) was used to perform the information processing.

Genotyping

Genomic SNP genotyping was performed using the Illumina^® Infinium HumanOmni5-4 v1.1 BeadChips (Illumina). SNP editing was on call rate (>99%) and minor allele frequency (>1%). Subjects with >2.5% of missing genotypes were discarded. A total of 47,360 markers were retained after edits.

Statistical analysis

The SNPs categorical data association analysis was performed by using Pearson's chi-square test to detect differences in genotypic frequencies. Bonferroni correction was used to reduce the chances of obtaining false-positive results (type I errors). A p-value of <5.0E-8 was the threshold for significance (before Bonferroni correction). Statistical analysis was performed using Ruby v2.2.2p85 with statsample (https://github.com/sciruby/statsample).

A paired t-test was used for within-pair analysis of methylation rate. Bonferroni correction was used to reduce the chances of obtaining false-positive results (type I errors). A p-value of <5.0E-7 was the threshold for significance (before Bonferroni correction). Statistical analysis was performed using R v3.1.2.

Results

Serum levels of TgAb

After setting a cutoff value for TgAb to >28 IU/mL (recommended cutoff value), 19 males (13.6% of all male) and 72 females (19.3% of all female) subjects were positive for TgAb. To exclude the subjects with marginally elevated levels and in order to perform stricter grouping, the cutoff value for positive TgAb was set to 50.0 IU/mL (TgAb negative: <28.0 IU/mL; TgAb positive: ≥50.0 IU/mL; TgAb borderline: ≥28.0 IU/mL and <50.0 IU/mL; Fig. 1). In order to avoid incorrect categorization of twin pairs due to small intra-individual variations of TgAb, subjects showing TGAb levels in the borderline zone were excluded. As shown in Figure 1, using these criteria, TgAb positive concordant (PC) pairs accounted for 5.7% (four pairs) and 9.6% (18 pairs) of male and female pairs, respectively. TgAb discordant (DC) pairs (one twin was positive and the co-twin was negative) accounted for 11.4% (eight pairs) and 8.0% (15 pairs) of male and female pairs, respectively. TgAb negative concordant (NC) pairs accounted for 78.6% (55 pairs) of male pairs and 74.3% (139 pairs) of female pairs. No significant differences were found in the frequencies of PC, NC, and DC pairs between male and female pairs. The male pairs were significantly older than female pairs in each of the PC, NC, and DC groups (Table 1). Female pairs in the PC group were older than those in the DC group (p < 0.05; Table 1). TgAb, thyrotropin (TSH), and free thyroxine (fT4) levels did not show sex-related differences in each of the PC, NC, and DC groups (Table 1). TSH and fT4 levels were not different between TgAb positive and negative twins in the DC group (Table 1). However, a few females in the NC group showed elevated TSH levels, probably due to reasons other than AITD.

FIG. 1.

The criteria to categorize twin subjects and the protocol of analysis.

Table 1.

Clinical Characteristics of Examined Monozygotic Twins

Group	Sex	Number of pair	Age	Sex-difference of age	TgAb (IU/mL)		TSH (μIU/mL)		fT4 (ng/mL)
Positive concordant	All	20	59.3 ± 15.5		55–2340 (387.1 ± 390.4)		2.90 ± 2.70		0.82 ± 0.12
	Male	3	76.0 ± 4.4	p < 0.05	72–957 (502.0 ± 305.2)		3.85 ± 3.77		0.80 ± 0.11
	Female	17	56.3 ± 14.9^*	p < 0.05	55–2340 (356.8 ± 408.0)		2.65 ± 2.34		0.83 ± 0.12
Negative concordant	All	165	51.8 ± 19.0		ND (<5) - 27		2.48 ± 5.82		0.84 ± 0.13
	Male	45	60.1 ± 18.9	p < 0.001	ND (<5) - 25		1.93 ± 1.41		0.86 ± 0.12
	Female	120	48.7 ± 18.2	p < 0.001	ND(<5) - 27		2.70 ± 6.81		0.83 ± 0.13
Discordant					Positive twin	Negative twin	Positive twin	Negative twin	Positive twin	Negative twin
	All	19	51.5 ± 15.6		59–866 (266.4 ± 224.5)	ND(<5) - 27	2.13 ± 1.19	2.16 ± 1.57	0.86 ± 0.19	0.87 ± 0.14
	Male	6	69.0 ± 4.5	p < 0.0001	59–413 (143.1 ± 123.2)	ND(<5) - 27	2.35 ± 1.51	2.51 ± 1.50	0.90 ± 0.26	0.83 ± 0.14
	Female	13	43.4 ± 11.5	p < 0.0001	60–866 (332.2 ± 241.5)	ND(<5) - 24	2.02 ± 1.03	1.97 ± 1.62	0.84 ± 0.15	0.90 ± 0.15

p < 0.05 vs. female of discordant group.

TgAb, antithyroglobulin autoantibodies; TSH, thyrotropin; fT4, free thyroxine; ND, Not detected.

Methylation levels of CpG sites in TgAb discordant pairs

As shown in Figure 2 and Supplementary Table S1 (Supplementary Data are available online at www.liebertpub.com/thy), within-pair differences analyzed by a paired t-test indicated that methylation levels of 12,901 CpG sites were significantly different between TgAb positive and negative individuals of the 19 TgAb discordant pairs. However, none of them were significant after correction for multiple comparisons.

FIG. 2.

The differences in methylation levels between twins positive for antithyroglobulin autoantibodies (TgAb) and co-twins negative for TgAb in discordant twin pairs. A paired t-test was used for within-pair analysis of methylation rate. Bonferroni correction was used to reduce the risk of obtaining false-positive results (type I errors). A p-value of <5.0E-7 was the threshold for significance (before Bonferroni correction). The horizontal line indicates a suggestive but not significant level (1.0E-5) of the p-value after Bonferroni correction.

Differences in distributions of genotypes between TgAb concordant and discordant pair groups

As shown in Figure 3A, the distributions of some genotypes, mainly in chromosomes 6 and 14, were significantly different between PC and NC pair groups (significant markers are summarized in Supplementary Table S2). On the other hand, as shown in Figure 3B and C, the distributions of some genotypes in other chromosomes were significantly different between NC and DC pair groups and between concordant (CC; PC and NC) and DC pair groups. As shown in Table 2, 155 polymorphisms showed significant differences in the frequencies of genotypes between in TgAb DC and CC pair groups after correction for multiple comparisons.

FIG. 3.

Manhattan plot of p-values in genome-wide association study between (A) groups of positive concordant and negative concordant twins, (B) groups of discordant and concordant (including both positive and negative) twins, and (C) groups of discordant and negative concordant twins. The SNP categorical data association analysis was performed by using Pearson's chi-square test to detect differences in genotypic frequencies. Bonferroni correction was used to reduce the risk of obtaining false-positive results (type I errors). A p-value of <5.0E-8 was the threshold for significance (before Bonferroni correction). Horizontal lines indicate significant (5.0E-8) and suggestive (1.0E-5) levels of the p-value after Bonferroni correction.

Table 2.

Frequencies of Genotypes in TgAb Concordant and Discordant Pair Groups

		Numbers and percentages of genotype holders in each pair
		Concordant pairs			Discordant pairs
SNP	Allele (major/minor)	Major homozygote	Heterozygote	Minor homozygote	Major homozygote	Heterozygote	Minor homozygote	p ^*	pc ^**
rs56211683	T/G	129 (71.27)	52 (28.73)	0 (0.00)	8 (42.11)	6 (31.58)	5 (26.32)	4.60E-11	2.18E-06
rs61864241	C/T	185 (100.00)	0 (0.00)	0 (0.00)	15 (78.95)	4 (21.05)	0 (0.00)	2.92E-10	1.38E-05
rs78946853	T/C	135 (72.97)	47 (25.41)	3 (1.62)	8 (44.44)	4 (22.22)	6 (33.33)	3.06E-10	1.45E-05
rs147009241	A/C	182 (98.38)	3 (1.62)	0 (0.00)	13 (68.42)	6 (31.58)	0 (0.00)	1.40E-09	6.63E-05
rs142708001	A/G	182 (98.38)	3 (1.62)	0 (0.00)	13 (68.42)	6 (31.58)	0 (0.00)	1.40E-09	6.63E-05
rs35707110	C/T	105 (56.76)	79 (42.70)	1 (0.54)	9 (47.37)	5 (26.32)	5 (26.32)	1.76E-09	8.33E-05
rs17229224	A/G	105 (56.76)	79 (42.70)	1 (0.54)	9 (47.37)	5 (26.32)	5 (26.32)	1.76E-09	8.33E-05
rs62385515	G/A	105 (56.76)	79 (42.70)	1 (0.54)	9 (47.37)	5 (26.32)	5 (26.32)	1.76E-09	8.33E-05
rs456366	T/C	145 (78.38)	40 (21.62)	0 (0.00)	13 (68.42)	2 (10.53)	4 (21.05)	1.87E-09	8.84E-05
rs31489	C/A	145 (78.38)	40 (21.62)	0 (0.00)	13 (68.42)	2 (10.53)	4 (21.05)	1.87E-09	8.84E-05
rs452384	A/G	145 (78.38)	40 (21.62)	0 (0.00)	13 (68.42)	2 (10.53)	4 (21.05)	1.87E-09	8.84E-05
rs452932	A/G	145 (78.38)	40 (21.62)	0 (0.00)	13 (68.42)	2 (10.53)	4 (21.05)	1.87E-09	8.84E-05
rs31490	G/A	146 (78.92)	39 (21.08)	0 (0.00)	13 (68.42)	2 (10.53)	4 (21.05)	1.92E-09	9.07E-05
rs414965	C/T	147 (79.46)	38 (20.54)	0 (0.00)	13 (68.42)	2 (10.53)	4 (21.05)	1.96E-09	9.30E-05
rs4975615	A/G	142 (76.76)	43 (23.24)	0 (0.00)	12 (63.16)	3 (15.79)	4 (21.05)	2.28E-09	0.0001082
rs4975616	A/G	142 (76.76)	43 (23.24)	0 (0.00)	12 (63.16)	3 (15.79)	4 (21.05)	2.28E-09	0.0001082
rs61574973	C/T	143 (77.30)	42 (22.70)	0 (0.00)	12 (63.16)	3 (15.79)	4 (21.05)	2.31E-09	0.0001093
rs10078017	T/C	143 (77.30)	42 (22.70)	0 (0.00)	12 (63.16)	3 (15.79)	4 (21.05)	2.31E-09	0.0001093
rs37008	C/T	152 (82.16)	33 (17.84)	0 (0.00)	12 (63.16)	3 (15.79)	4 (21.05)	2.32E-09	0.0001099
rs37006	G/A	148 (80.00)	37 (20.00)	0 (0.00)	12 (63.16)	3 (15.79)	4 (21.05)	2.36E-09	0.0001119
rs27069	G/A	148 (80.43)	36 (19.57)	0 (0.00)	12 (63.16)	3 (15.79)	4 (21.05)	1.18E-08	0.0005578
rs7582260	A/G	119 (64.32)	64 (34.59)	2 (1.08)	12 (63.16)	2 (10.53)	5 (26.32)	2.43E-08	0.0011513
rs116808972	T/C	150 (81.97)	33 (18.03)	0 (0.00)	8 (42.11)	8 (42.11)	3 (15.79)	3.05E-08	0.0014442
rs72854540	C/T	184 (99.46)	1 (0.54)	0 (0.00)	15 (78.95)	4 (21.05)	0 (0.00)	3.66E-08	0.0017335
rs56107673	C/T	184 (99.46)	1 (0.54)	0 (0.00)	15 (78.95)	4 (21.05)	0 (0.00)	3.66E-08	0.0017335
rs78563513	G/A	184 (99.46)	1 (0.54)	0 (0.00)	15 (78.95)	4 (21.05)	0 (0.00)	3.66E-08	0.0017335
rs117855341	G/T	185 (100.00)	0 (0.00)	0 (0.00)	16 (84.21)	3 (15.79)	0 (0.00)	5.18E-08	0.0024554
rs143039049	G/A	185 (100.00)	0 (0.00)	0 (0.00)	16 (84.21)	3 (15.79)	0 (0.00)	5.18E-08	0.0024554
rs118046582	T/G	185 (100.00)	0 (0.00)	0 (0.00)	16 (84.21)	3 (15.79)	0 (0.00)	5.18E-08	0.0024554
rs11923450	A/G	185 (100.00)	0 (0.00)	0 (0.00)	16 (84.21)	3 (15.79)	0 (0.00)	5.18E-08	0.0024554
rs72922940	A/G	185 (100.00)	0 (0.00)	0 (0.00)	16 (84.21)	3 (15.79)	0 (0.00)	5.18E-08	0.0024554
rs1079464	G/A	116 (62.70)	67 (36.22)	2 (1.08)	10 (52.63)	4 (21.05)	5 (26.32)	5.60E-08	0.0026503
rs17613848	G/A	131 (70.81)	52 (28.11)	2 (1.08)	9 (47.37)	5 (26.32)	5 (26.32)	5.66E-08	0.0026792
rs745693	G/A	117 (63.24)	66 (35.68)	2 (1.08)	10 (52.63)	4 (21.05)	5 (26.32)	5.70E-08	0.0026982
rs117045501	A/G	175 (94.59)	10 (5.41)	0 (0.00)	11 (57.89)	8 (42.11)	0 (0.00)	7.83E-08	0.0037098
rs117108505	A/G	177 (96.72)	6 (3.28)	0 (0.00)	12 (63.16)	7 (36.84)	0 (0.00)	7.95E-08	0.0037647
rs60924571	G/A	123 (66.49)	58 (31.35)	4 (2.16)	9 (47.37)	4 (21.05)	6 (31.58)	1.13E-07	0.0053522
rs6580584	T/C	128 (69.19)	56 (30.27)	1 (0.54)	13 (68.42)	2 (10.53)	4 (21.05)	1.16E-07	0.0054855
rs389694	G/T	150 (81.08)	34 (18.38)	1 (0.54)	10 (52.63)	5 (26.32)	4 (21.05)	1.17E-07	0.0055351
rs10182999	C/T	120 (65.22)	62 (33.70)	2 (1.09)	12 (63.16)	2 (10.53)	5 (26.32)	1.23E-07	0.0058018
rs6769725	C/T	129 (69.73)	55 (29.73)	1 (0.54)	8 (42.11)	7 (36.84)	4 (21.05)	1.23E-07	0.0058225
rs74019045	G/A	182 (98.38)	3 (1.62)	0 (0.00)	14 (73.68)	5 (26.32)	0 (0.00)	1.29E-07	0.0060917
rs12596647	G/T	182 (98.38)	3 (1.62)	0 (0.00)	14 (73.68)	5 (26.32)	0 (0.00)	1.29E-07	0.0060917
rs7290176	G/A	86 (46.49)	87 (47.03)	12 (6.49)	4 (21.05)	6 (31.58)	9 (47.37)	1.52E-07	0.0071824
rs792599	A/C	130 (70.65)	46 (25.00)	8 (4.35)	7 (36.84)	4 (21.05)	8 (42.11)	1.61E-07	0.0076126
rs56102538	A/G	134 (72.43)	50 (27.03)	1 (0.54)	9 (47.37)	6 (31.58)	4 (21.05)	1.64E-07	0.0077444
rs6787392	A/G	150 (81.08)	35 (18.92)	0 (0.00)	15 (78.95)	1 (5.26)	3 (15.79)	1.82E-07	0.0086089
rs1073406	C/T	142 (76.76)	42 (22.70)	1 (0.54)	10 (52.63)	5 (26.32)	4 (21.05)	1.84E-07	0.008691
rs9342183	C/T	123 (66.49)	62 (33.51)	0 (0.00)	13 (68.42)	3 (15.79)	3 (15.79)	1.93E-07	0.0091504
rs849000	T/C	152 (82.16)	33 (17.84)	0 (0.00)	15 (78.95)	1 (5.26)	3 (15.79)	1.98E-07	0.0093914
rs9362624	G/T	124 (67.03)	61 (32.97)	0 (0.00)	13 (68.42)	3 (15.79)	3 (15.79)	2.01E-07	0.0095323
rs9344911	T/C	124 (67.03)	61 (32.97)	0 (0.00)	13 (68.42)	3 (15.79)	3 (15.79)	2.01E-07	0.0095323
rs321206	C/G	158 (85.87)	25 (13.59)	1 (0.54)	10 (52.63)	5 (26.32)	4 (21.05)	2.13E-07	0.0100983
rs72985433	A/G	118 (63.78)	67 (36.22)	0 (0.00)	8 (42.11)	8 (42.11)	3 (15.79)	2.17E-07	0.0102907
rs415026	G/A	148 (80.00)	36 (19.46)	1 (0.54)	11 (57.89)	4 (21.05)	4 (21.05)	2.18E-07	0.0103068
rs710511	C/T	155 (83.87)	30 (16.22)	0 (0.00)	15 (78.95)	1 (5.26)	3 (15.79)	2.24E-07	0.0106301
rs710512	G/T	155 (83.87)	30 (16.22)	0 (0.00)	15 (78.95)	1 (5.26)	3 (15.79)	2.24E-07	0.0106301
rs321223	C/T	144 (77.84)	40 (21.62)	1 (0.54)	11 (57.89)	4 (21.05)	4 (21.05)	2.40E-07	0.0113794
rs6475418	A/G	144 (77.84)	40 (21.62)	1 (0.54)	11 (57.89)	4 (21.05)	4 (21.05)	2.40E-07	0.0113794
rs7036564	G/A	144 (77.84)	40 (21.62)	1 (0.54)	11 (57.89)	4 (21.05)	4 (21.05)	2.40E-07	0.0113794
rs321225	G/A	144 (77.84)	40 (21.62)	1 (0.54)	11 (57.89)	4 (21.05)	4 (21.05)	2.40E-07	0.0113794
rs117145089	T/A	174 (94.05)	11 (5.95)	0 (0.00)	11 (57.89)	8 (42.11)	0 (0.00)	2.41E-07	0.0114126
rs78149682	T/G	174 (94.05)	11 (5.95)	0 (0.00)	11 (57.89)	8 (42.11)	0 (0.00)	2.41E-07	0.0114126
rs17087180	A/G	156 (84.32)	29 (15.68)	0 (0.00)	12 (63.16)	4 (21.05)	3 (15.79)	2.44E-07	0.0115347
rs75379370	A/G	158 (85.41)	27 (14.59)	0 (0.00)	15 (78.95)	1 (5.26)	3 (15.79)	2.52E-07	0.0119305
rs421284	T/C	142 (76.76)	42 (22.70)	1 (0.54)	12 (63.16)	3 (15.79)	4 (21.05)	2.54E-07	0.0120175
rs465498	T/C	142 (76.76)	42 (22.70)	1 (0.54)	12 (63.16)	3 (15.79)	4 (21.05)	2.54E-07	0.0120175
rs148609907	G/A	177 (95.68)	8 (4.32)	0 (0.00)	12 (63.16)	6 (31.58)	1 (5.26)	2.56E-07	0.0121414
rs6744298	T/G	131 (70.81)	53 (28.65)	1 (0.54)	11 (57.89)	4 (21.05)	4 (21.05)	2.57E-07	0.0121787
rs32757	A/G	131 (70.81)	51 (27.57)	3 (1.62)	7 (36.84)	7 (36.84)	5 (26.32)	2.69E-07	0.0127629
rs12139436	C/A	154 (83.24)	31 (16.76)	0 (0.00)	12 (63.16)	4 (21.05)	3 (15.79)	2.70E-07	0.0127846
rs2294944	C/G	154 (83.24)	31 (16.76)	0 (0.00)	12 (63.16)	4 (21.05)	3 (15.79)	2.70E-07	0.0127846
rs12166240	A/G	154 (83.24)	31 (16.76)	0 (0.00)	12 (63.16)	4 (21.05)	3 (15.79)	2.70E-07	0.0127846
rs16862632	T/G	154 (83.24)	31 (16.76)	0 (0.00)	12 (63.16)	4 (21.05)	3 (15.79)	2.70E-07	0.0127846
rs17159072	C/T	154 (83.24)	31 (16.76)	0 (0.00)	12 (63.16)	4 (21.05)	3 (15.79)	2.70E-07	0.0127846
rs3917205	C/T	147 (79.46)	38 (20.54)	0 (0.00)	14 (73.68)	2 (10.53)	3 (15.79)	2.82E-07	0.013343
rs7927239	G/C	179 (100.00)	0 (0.00)	0 (0.00)	15 (83.33)	3 (16.67)	0 (0.00)	2.89E-07	0.0137089
rs1005793	T/C	164 (88.65)	21 (11.35)	0 (0.00)	15 (78.95)	1 (5.26)	3 (15.79)	3.08E-07	0.014578
rs17070841	T/C	164 (88.65)	21 (11.35)	0 (0.00)	15 (78.95)	1 (5.26)	3 (15.79)	3.08E-07	0.014578
rs17070861	T/G	164 (88.65)	21 (11.35)	0 (0.00)	15 (78.95)	1 (5.26)	3 (15.79)	3.08E-07	0.014578
rs7076675	A/G	129 (69.73)	56 (30.27)	0 (0.00)	10 (52.63)	6 (31.58)	3 (15.79)	3.12E-07	0.0147838
rs8135478	T/C	138 (74.59)	47 (25.41)	0 (0.00)	13 (68.42)	3 (15.79)	3 (15.79)	3.14E-07	0.0148596
rs10503079	C/T	165 (89.19)	20 (10.81)	0 (0.00)	15 (78.95)	1 (5.26)	3 (15.79)	3.17E-07	0.0149998
rs7290267	A/G	139 (75.14)	46 (24.86)	0 (0.00)	13 (68.42)	3 (15.79)	3 (15.79)	3.21E-07	0.0151878
rs9842506	G/T	152 (82.16)	33 (17.84)	0 (0.00)	14 (73.68)	2 (10.53)	3 (15.79)	3.22E-07	0.0152334
rs2274000	T/C	159 (85.95)	26 (14.05)	0 (0.00)	13 (68.42)	3 (15.79)	3 (15.79)	3.26E-07	0.0154245
rs9626891	T/C	140 (75.68)	45 (24.32)	0 (0.00)	13 (68.42)	3 (15.79)	3 (15.79)	3.27E-07	0.0155002
(kgp15464535)	G/A	178 (100.00)	0 (0.00)	0 (0.00)	15 (83.33)	3 (16.67)	0 (0.00)	3.30E-07	0.0156174
rs75568212	C/T	141 (76.22)	44 (23.78)	0 (0.00)	13 (68.42)	3 (15.79)	3 (15.79)	3.34E-07	0.0157951
rs4660123	C/A	158 (85.41)	27 (14.59)	0 (0.00)	13 (68.42)	3 (15.79)	3 (15.79)	3.35E-07	0.0158502
rs6691710	G/T	158 (85.41)	27 (14.59)	0 (0.00)	13 (68.42)	3 (15.79)	3 (15.79)	3.35E-07	0.0158502
rs7775554	C/A	129 (69.73)	56 (30.27)	0 (0.00)	12 (63.16)	4 (21.05)	3 (15.79)	3.35E-07	0.0158841
rs34855929	A/G	183 (100.00)	0 (0.00)	0 (0.00)	16 (84.21)	3 (15.79)	0 (0.00)	3.38E-07	0.0160268
rs2241999	C/T	156 (84.32)	29 (15.68)	0 (0.00)	14 (73.68)	2 (10.53)	3 (15.79)	3.47E-07	0.0164554
rs79802155	T/C	169 (91.35)	16 (8.65)	0 (0.00)	15 (78.95)	1 (5.26)	3 (15.79)	3.48E-07	0.016487
rs117423166	C/A	169 (91.35)	16 (8.65)	0 (0.00)	15 (78.95)	1 (5.26)	3 (15.79)	3.48E-07	0.016487
rs754215	G/A	156 (84.32)	29 (15.68)	0 (0.00)	13 (68.42)	3 (15.79)	3 (15.79)	3.49E-07	0.0165261
rs12124410	T/C	156 (84.32)	29 (15.68)	0 (0.00)	13 (68.42)	3 (15.79)	3 (15.79)	3.49E-07	0.0165261
rs77688708	C/A	184 (99.46)	0 (0.00)	1 (0.54)	16 (84.21)	3 (15.79)	0 (0.00)	3.52E-07	0.0166488
rs150047065	C/T	184 (100.00)	0 (0.00)	0 (0.00)	16 (84.21)	3 (15.79)	0 (0.00)	3.52E-07	0.0166488
rs78221722	C/T	184 (100.00)	0 (0.00)	0 (0.00)	16 (84.21)	3 (15.79)	0 (0.00)	3.52E-07	0.0166488
rs12254500	C/T	133 (71.89)	52 (28.11)	0 (0.00)	11 (57.89)	5 (26.32)	3 (15.79)	3.54E-07	0.0167601
rs73471350	C/A	142 (76.76)	43 (23.24)	0 (0.00)	12 (63.16)	4 (21.05)	3 (15.79)	3.61E-07	0.0170992
rs2296974	G/A	142 (76.76)	43 (23.24)	0 (0.00)	12 (63.16)	4 (21.05)	3 (15.79)	3.61E-07	0.0170992
rs10485251	T/G	142 (76.76)	43 (23.24)	0 (0.00)	12 (63.16)	4 (21.05)	3 (15.79)	3.61E-07	0.0170992
rs9812017	C/A	153 (82.70)	32 (17.30)	0 (0.00)	13 (68.42)	3 (15.79)	3 (15.79)	3.62E-07	0.0171271
rs17194133	G/T	152 (82.16)	33 (17.84)	0 (0.00)	13 (68.42)	3 (15.79)	3 (15.79)	3.64E-07	0.017226
rs59555562	C/T	129 (69.73)	56 (30.27)	0 (0.00)	11 (57.89)	5 (26.32)	3 (15.79)	3.64E-07	0.0172383
rs112085050	A/T	151 (81.62)	34 (18.38)	0 (0.00)	13 (68.42)	3 (15.79)	3 (15.79)	3.65E-07	0.0172783
rs59526446	C/T	128 (69.19)	57 (30.81)	0 (0.00)	11 (57.89)	5 (26.32)	3 (15.79)	3.65E-07	0.0172784
rs3845675	C/T	128 (69.19)	57 (30.81)	0 (0.00)	11 (57.89)	5 (26.32)	3 (15.79)	3.65E-07	0.0172784
rs13417979	A/C	128 (69.19)	57 (30.81)	0 (0.00)	11 (57.89)	5 (26.32)	3 (15.79)	3.65E-07	0.0172784
rs8060152	C/G	138 (74.59)	47 (25.41)	0 (0.00)	12 (63.16)	4 (21.05)	3 (15.79)	3.65E-07	0.0172788
rs10951647	T/C	138 (74.59)	47 (25.41)	0 (0.00)	12 (63.16)	4 (21.05)	3 (15.79)	3.65E-07	0.0172788
rs255013	T/C	138 (74.59)	47 (25.41)	0 (0.00)	12 (63.16)	4 (21.05)	3 (15.79)	3.65E-07	0.0172788
rs11887482	G/A	127 (68.65)	58 (31.35)	0 (0.00)	11 (57.89)	5 (26.32)	3 (15.79)	3.65E-07	0.0172879
rs4060593	A/G	127 (68.65)	58 (31.35)	0 (0.00)	11 (57.89)	5 (26.32)	3 (15.79)	3.65E-07	0.0172879
rs115833701	A/G	163 (88.11)	22 (11.89)	0 (0.00)	10 (52.63)	7 (36.84)	2 (10.53)	3.78E-07	0.0179074
rs12882780	G/T	174 (94.05)	10 (5.41)	1 (0.54)	11 (57.89)	8 (42.11)	0 (0.00)	5.33E-07	0.0252552
rs62139469	G/T	133 (71.89)	47 (25.41)	5 (2.70)	8 (42.11)	5 (26.32)	6 (31.58)	5.40E-07	0.0255882
rs56239333	G/T	126 (68.48)	58 (31.52)	0 (0.00)	14 (73.68)	2 (10.53)	3 (15.79)	5.41E-07	0.0256178
rs4943880	T/C	97 (52.43)	71 (38.38)	17 (9.19)	4 (21.05)	5 (26.32)	10 (52.63)	5.95E-07	0.0281853
rs2471827	C/T	108 (60.34)	68 (37.99)	3 (1.68)	9 (52.94)	3 (17.65)	5 (29.41)	6.16E-07	0.0291689
rs677079	C/A	155 (83.78)	30 (16.22)	0 (0.00)	9 (47.37)	8 (42.11)	2 (10.53)	6.43E-07	0.0304317
rs1473254	T/C	161 (87.03)	24 (12.97)	0 (0.00)	10 (52.63)	7 (36.84)	2 (10.53)	6.93E-07	0.0328025
rs1473256	G/A	161 (87.03)	24 (12.97)	0 (0.00)	10 (52.63)	7 (36.84)	2 (10.53)	6.93E-07	0.0328025
rs1549950	A/G	161 (87.03)	24 (12.97)	0 (0.00)	10 (52.63)	7 (36.84)	2 (10.53)	6.93E-07	0.0328025
rs4804941	A/G	161 (87.03)	24 (12.97)	0 (0.00)	10 (52.63)	7 (36.84)	2 (10.53)	6.93E-07	0.0328025
rs56154743	C/A	132 (71.35)	50 (27.03)	3 (1.68)	9 (47.37)	5 (26.32)	5 (26.32)	7.41E-07	0.0350982
rs3761137	G/A	132 (71.35)	50 (27.03)	3 (1.68)	9 (47.37)	5 (26.32)	5 (26.32)	7.41E-07	0.0350982
rs55956760	A/G	176 (95.14)	9 (4.86)	0 (0.00)	12 (63.16)	7 (36.84)	0 (0.00)	7.93E-07	0.0375358
rs55914918	C/T	118 (63.78)	64 (34.59)	3 (1.62)	10 (52.63)	4 (21.05)	5 (26.32)	7.98E-07	0.037787
rs855447	T/C	108 (58.38)	74 (40.00)	3 (1.68)	9 (47.37)	5 (26.32)	5 (26.32)	8.35E-07	0.0395682
rs855530	C/T	109 (58.92)	73 (39.46)	3 (1.68)	9 (47.37)	5 (26.32)	5 (26.32)	8.44E-07	0.0399944
(kgp4640232)	T/C	163 (90.06)	18 (9.94)	0 (0.00)	13 (68.42)	3 (15.79)	3 (15.79)	8.46E-07	0.040063
rs4977391	G/A	124 (67.03)	58 (31.35)	3 (1.62)	10 (52.63)	4 (21.05)	5 (26.32)	8.58E-07	0.0406196
rs1487434	A/G	153 (82.70)	32 (17.30)	0 (0.00)	7 (36.84)	11 (57.89)	1 (5.26)	9.05E-07	0.0428754
rs78459177	C/T	183 (98.92)	2 (1.08)	0 (0.00)	15 (78.95)	4 (21.05)	0 (0.00)	9.27E-07	0.0438863
rs117456519	G/T	183 (98.92)	2 (1.08)	0 (0.00)	15 (78.95)	4 (21.05)	0 (0.00)	9.27E-07	0.0438863
rs75994098	A/G	183 (98.92)	2 (1.08)	0 (0.00)	15 (78.95)	4 (21.05)	0 (0.00)	9.27E-07	0.0438863
rs11684191	C/T	183 (98.92)	2 (1.08)	0 (0.00)	15 (78.95)	4 (21.05)	0 (0.00)	9.27E-07	0.0438863
rs143197066	C/T	183 (98.92)	2 (1.08)	0 (0.00)	15 (78.95)	4 (21.05)	0 (0.00)	9.27E-07	0.0438863
rs62070169	A/C	183 (98.92)	2 (1.08)	0 (0.00)	15 (78.95)	4 (21.05)	0 (0.00)	9.27E-07	0.0438863
rs3741450	G/T	183 (98.92)	2 (1.08)	0 (0.00)	15 (78.95)	4 (21.05)	0 (0.00)	9.27E-07	0.0438863
rs57512987	G/A	183 (98.92)	2 (1.08)	0 (0.00)	15 (78.95)	4 (21.05)	0 (0.00)	9.27E-07	0.0438863
rs17007760	G/A	183 (98.92)	2 (1.08)	0 (0.00)	15 (78.95)	4 (21.05)	0 (0.00)	9.27E-07	0.0438863
rs890667	G/A	183 (98.92)	2 (1.08)	0 (0.00)	15 (78.95)	4 (21.05)	0 (0.00)	9.27E-07	0.0438863
rs3741447	G/A	183 (98.92)	2 (1.08)	0 (0.00)	15 (78.95)	4 (21.05)	0 (0.00)	9.27E-07	0.0438863
rs2516087	G/T	79 (42.70)	92 (49.73)	14 (7.57)	6 (31.58)	4 (21.05)	9 (47.37)	9.51E-07	0.0450262
rs360777	T/C	181 (97.84)	4 (2.16)	0 (0.00)	14 (73.68)	5 (26.32)	0 (0.00)	1.05E-06	0.0496678
rs79512588	G/A	181 (97.84)	4 (2.16)	0 (0.00)	14 (73.68)	5 (26.32)	0 (0.00)	1.05E-06	0.0496678
rs142441049	G/A	181 (97.84)	4 (2.16)	0 (0.00)	14 (73.68)	5 (26.32)	0 (0.00)	1.05E-06	0.0496678
rs11608863	T/C	181 (97.84)	4 (2.16)	0 (0.00)	14 (73.68)	5 (26.32)	0 (0.00)	1.05E-06	0.0496678
rs9933397	C/T	181 (97.84)	4 (2.16)	0 (0.00)	14 (73.68)	5 (26.32)	0 (0.00)	1.05E-06	0.0496678
rs11016258	C/T	181 (97.84)	4 (2.16)	0 (0.00)	14 (73.68)	5 (26.32)	0 (0.00)	1.05E-06	0.0496678

SNP names in parentheses are code used in Illumina kit. SNP used to analyze the genotype-specific methylation are indicated in bold.

p-Value tested by chi-square test; ^**corrected p-value by Bonferroni's correction.

Methylation levels of CpG sites in genotype-based TgAb discordant pairs

Among 155 polymorphisms showing significant differences in the genotype frequencies between TgAb DC and CC pairs (Table 2), the study focused on 10 polymorphisms with the following criteria: (i) polymorphisms must show the typical difference between DC and CC pairs, (ii) sample number of the specific genotype for the DC pair is enough to analyze, and (iii) one representative polymorphism among linked polymorphisms in each region must be selected.

In DC pairs with DC-specific genotypes of each of the representative polymorphism, within-pair differences in methylation levels of each CpG site were compared using a paired t-test (Supplementary Table S3). Among these representative polymorphisms, significant differences were found in DNA methylation levels of CpG sites between TgAb positive and negative twins of DC pairs with five specific polymorphisms (rs78946853T/C, rs56211683T/G, rs35707110C/T, rs31489C/A, and rs7290176G/A). In the DC pairs with rs35707110TT, there was a significant CpG site (cg10261589; p = 0). Because DNA methylation of this CpG site we could only be evaluated in two pairs, however, this site was excluded to increase reliability.

As shown in Figure 4 and Table 3, methylation levels of four CpG sites (cg17082856 in rs78946853CC, cg23898213 in rs56211683GG, cg00334293 in rs31489AA, and cg14990605 in rs7290176AA) were significantly different after correction for multiple comparisons between TgAb positive and negative co-twins within the DC-specific genotype. Figure 5 shows the parallel plots of methylation levels of these four CpG sites between the TgAb positive twin and the negative co-twin categorized into three DC-specific polymorphisms.

FIG. 4.

Manhattan plot of p-values in paired t-tests of differences in genotype-specific methylation levels between twins positive for TgAb and co-twins negative for TgAb in discordant twin pairs. (A) cg17082856 site in rs78946853 CC genotype, (B) cg23898213 site in rs56211683 GG genotype, (C) cg00334293 site in rs31489 AAgenotype, and (D) cg14990605 site in rs7290176 AA genotype. A paired t-test was used for within-pair analysis of methylation rate. Bonferroni correction was used to reduce the risk of obtaining false-positive results (type I errors). A p-value of <5.0E-7 was the threshold for significance (before Bonferroni correction). Horizontal lines indicate significant (5.0E-7) and suggestive (1.0E-4) levels of the p-value after Bonferroni correction.

FIG. 5.

Parallel plots of genotype-specific methylation levels between twins positive for TgAb and co-twins negative for TgAb in discordant twin pairs. (A) cg17082856 site in rs78946853 CC genotype, (B) cg23898213 site in rs56211683 GG genotype, (C) cg00334293 site in rs31489 AA genotype, and (D) cg14990605 site in rs7290176 AA genotype.

Table 3.

CpG Sites with Significant Differences in DNA Methylation Levels Between TgAb Positive and Negative Twins of TgAb Discordant Pairs with TgAb-Specific Polymorphisms

SNP for grouping			Compared CpG sites			p-Value of paired t-test in TgAb discordant twins with:
rs No.	Chromosome	Gene	Name	Chromosome	Gene	Group	Non-specific homozygote	Specific homozygote	Non-specific allele carrier	Specific allele carrier
rs78946853	2	CAPN13	cg17082856	2	CEP68	Body	0.27471980	0.00000047	0.23425956	0.01129082
rs56211683	20	Near LOC105372683	cg23898213	X			0.11714652	0.00000033	0.93833408	0.04860348
rs31489	5	CLPTM1L	cg00334293	22			0.37635819	0.00000076	0.37635819	0.00000076
rs7290176	22	LARGE1	cg14990605	10			0.05447372	0.00000017	0.24448518	0.87492856

Significant p-values after correction for multi-comparison are shown in bold.

Discussion

Using recommended cutoff values for TgAb, the prevalence of TgAb in this study (13.6% of all male and 19.3% of all women) was slightly different from a previous report in Japanese (13.1% in male and 27.4% in female subjects) (16), probably because of differences in the method to measure the antibodies (ECLIA in this study vs. radioimmunoassay in the previous study).

In the present study, methylation levels of many CpG sites showed significant associations with the appearance of thyroid-specific autoantibodies (Fig. 2 and Supplementary Table S1). However, these associations were not statistically significant after multiple comparisons. It was supposed that the effects of DNA methylation would vary based on genetic background; that is, some DNA methylation in individuals with a specific genetic background will affect the development of autoantibodies, whereas those with another genetic background will not. Therefore, the study tried to determine significant genetic differences between TgAb concordant and discordant pairs to find specific genotypes for discordant pairs. As shown in Table 2 and Figure 3C, 155 polymorphisms were found showing significant differences in the genotype frequencies between TgAb DC and CC pairs. These polymorphisms were located in a large variety of genes (Table 2), and these results show specific genotypes for TgAb discordant pairs. These data suggest that some individuals may have a genetic susceptibility to epigenetic or environmental factors for autoantibody production, whereas others may be resistant to developing them.

Interestingly, chromosomes containing these significant polymorphisms were not always the same as those containing established AITD susceptibility genes (Fig. 3A, such as HLA located on chromosome 6) (1). This indicates that the genes related to genetic and epigenetic susceptibility to thyroid autoimmunity are different. Supporting this, as shown in Figure 3B, the chromosomes with significant polymorphisms between NC and DC were not always located on chromosomes containing AITD susceptibility genes. These data suggest that the balance among genetic susceptibility to TgAb production, genetic susceptibilities to epigenetic changes, and environmental factors affecting epigenetic changes may be independently related to the development of TgAb.

Next, the methylation levels of each CpG site were compared between TgAb positive and TgAb negative twins of each TgAb discordant pair with five specific genotypes for TgAb discordant pairs (rs78946853CC, rs56211683GG, rs35707110TT, rs31489AA, and rs7290176AA). As shown in Figure 4 and Table 3, four CpG sites showed significant differences between TgAb positive and negative twins in discordant pairs. As shown in Figure 5, methylation levels of cg17082856 were significantly higher in the TgAb positive twin than in the TgAb negative twin of discordant pairs with the rs78946853CC genotype. cg17082856 is located on the CEP68 gene and rs78946853 is located on the CAPN13 gene, and both genes are mapped on chromosome 2. CEP68 has been reported to be associated with acute urticaria/angioedema and a risk of aspirin-intolerant asthma (17,18), but no studies suggesting a relation of this gene with thyroid disorders or immune functions could be found. The CAPN13 gene, which is part of a family of cytosolic calcium-activated proteases involved in apoptosis, cell division, modulation of integrin-cytoskeletal interactions, and synaptic plasticity (19), is associated with hypertension (20). However, there is no report showing an association between CAPN13 and autoimmune disorders. In addition, no epistatic interactions between the CEP68 and CAPN13 genes resulting in an impact on autoimmunity have been reported.

Other significant CpG sites are cg23898213 in rs56211683GG, cg00334293 in rs31489AA, and cg14990605 in rs7290176AA. The genes containing each of these CpG sites and the genes containing each specific polymorphism are not mapped to the same chromosomes, as shown in Table 3. These data suggest that epigenetic changes may not always be affected by genetic factors on the same chromosome. Some of the genes associating with these polymorphisms are CLPTM1 L and LARGE1. CLIPTM1 L encodes a membrane protein, and polymorphisms in this gene have been reported to increase susceptibility to several cancers, including lung and pancreatic cancers (21,22). LARGE1 encodes a member of the N-acetylglucosaminyltransferase gene family, and mutations of this gene cause congenital muscular dystrophy with severe mental retardation (23). However, no reports of these genes showing associations with autoantibody production could be found. Moreover, it is not possible to comment on the effect of epigenetic changes in this gene because the region containing these specific CpG sites was not close to any novel genes, which is a limitation of this study. In addition, more samples are desirable to corroborate the findings of the analysis.

In conclusion, this twin study provides some insights into the genetic background underlying the epigenetic susceptibility for developing thyroid autoimmunity. Several coding or non-coding DNA regions may be associated with the production of thyroid-specific autoantibodies through epigenetic changes based on specific genetic background. However, the effects of these epigenetic changes on gene expression must be confirmed.

Footnotes

Acknowledgments

The authors are thankful to all the consultants from the Osaka Twin Research Group (Yoshinori Iwatani [lead author: iwatani@sahs.med.osaka-u.ac.jp], Norio Sakai, Hiroyasu Iso, Kei Kamide, Jun Hatazawa, Shinji Kihara, Hiroko Watanabe, Kiyoko Makimoto, Mikio Watanabe, and Chika Honda, the Center for Twin Research, Osaka University Graduate School of Medicine) and all the technical and secretarial staff of the Center for Twin Research, Osaka University Graduate School of Medicine. This project was supported by University Grants from the Japanese Ministry of Education, Culture, Sports, Science and Technology, and by JSPS KAKENHI JP16H03261. The authors are also thankful to Beckman Coulter, Inc. (Tokyo, Japan) for collaborative studies.

Author Disclosure Statement

The authors report no conflicts of interest in this work.

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