Abstract
Introduction: We have reported that high total homocysteine and the coexistence of inadequate thyroid hormones in maternal serum increase the risk of fetal neural tube defects (NTDs). Placental iodothyronine deiodinases (DIOs: DIO1, DIO2, and DIO3) play a role in regulating the conversions between different forms of maternal thyroid hormones. This study hypothesized that single nucleotide polymorphisms (SNPs) in placental DIOs genes could be related to NTDs. Methods: We performed a case-control study from 2007 to 2009 that included pregnant women from Lüliang, Shanxi Province, China. Nine distinct SNPs in DIOs genes were analyzed, and placental samples were obtained from 83 pregnant women with NTD fetuses and 90 pregnant women with normal fetuses. The nine SNPs were analyzed using the Cochran-Armitage test and the Fisher’s exact test. Results: There were no statistically significant differences between case and control in the nine SNPs of DIOs (p > 0.05). Conclusions: The results of this study suggested that SNPs of DIO genes in the placenta among pregnant women have no statistically significant difference between the two groups, suggesting that other factors might be involved in metabolism of maternal thyroid hormone provided to fetuses, such as epigenetic modification of methylation and homocysteinylation and genomic imprinting in the placenta. Further functional studies on placenta samples are necessary.
Introduction
Neural tube defects (NTDs) are severe birth defects of the central nervous system. A particularly high prevalence of NTDs, along with other congenital malformations, was recorded in 2003 in the Lüliang area, Shanxi Province, northern China [1, 2]. Our previous studies found that several risk factors are associated with NTDs, such as deficiency of folic acid and/or other micronutrients, high total homocysteine (tHCY) levels, and the coexistence of inadequate maternal serum levels of thyroid hormones which increase the risk of elevated tHCY in this area [3‒5].
Normally, thyroid hormones are essential for early fetal neurogenesis. Maternal thyroxine (T4) has been observed in embryonic circulation throughout gestation and as early as the fourth week of pregnancy [6]. Moreover, there is a peak of maternal T4 that results from high placental chorionic gonadotropin levels at approximately 10–12 gestational weeks [7]. The fetal thyroid gland reaches maturity by weeks 11 or 12 and begins to secrete thyroid hormones by approximately week 16 [6]. The presence of iodothyronine deiodinase type 3 (DIO3) in the placenta, uterus, and some fetal tissues is critical for minimizing the exposure of fetal tissues to inappropriate levels of thyroid hormone [8]. However, locally generated tri-iodothyronine (T3) in the brain from maternally transported T4 has been reported to be essential for normal early brain development [6, 9]. Almost 80% of neural T3 is produced locally by iodothyronine deiodinase type 2 (DIO2) [10]. The iodothyronine deiodinases (DIOs) (type 1, type 2, and type 3 [DIO1, DIO2, and DIO3, respectively]) constitute a potent mechanism of thyroid hormone activation (DIO1 and DIO2) or inactivation (DIO3). The predominant deiodinase expressed in the placenta is DIO3; however, DIO2 is also present [11]. It has been reported that some haplotype combinations of rs225010, rs225012, and rs1388378 were associated with mental retardation in iodine-deficient areas of China [12, 13]. With reference to this report, fetuses of those pregnant women who had both high tHCY and inadequate free T4 were 3 times more at risk (vs. controls) of developing NTDs in our previous study [5], and there was no association between NTDs and single nucleotide polymorphisms (SNPs) using maternal blood samples (rs11206237 and rs2235544 in the DIO1 gene; rs225010, rs225011, rs225012, rs225014, rs12885300, and rs1388378 in the DIO2 gene; and rs17716499 in the DIO3 gene) [5]. Therefore, we hypothesized that SNPs in these placental DIO1, DIO2, and DIO3 genes may be associated with fetal NTDs. This study was performed to test this hypothesis in placental samples.
Materials and Methods
Study Design
We performed a case-control study from 2007 to 2009 consisting of pregnant women from the Lüliang area, Shanxi Province, China, as previously reported [5].
Participants, Recruitment, and Diagnostic Criteria of NTDs (Outcome Measures)
Participants were those described in our previous study [5]. The median ages (minimum-maximum) of the mothers who had NTD fetuses and controls were 23 (19–41, n = 83) and 25 (18–41, n = 90) years old, respectively [5]. The frequency of participants positive for two anti-thyroid antibodies (anti-thyroid peroxidase antibody and anti-thyroglobulin antibody) did not differ significantly between the two groups.
NTDs in fetuses were diagnosed by B-mode ultrasound, which was conducted as a part of a routine health checkup at or from 12 gestational weeks in several local county hospitals. When NTDs were diagnosed by B-mode ultrasound, medical abortions were performed at the local county hospitals. Medical staff collected clinical information including gestational age in weeks and serum and spot urinary samples. Fetal samples from women who underwent pregnancy termination for nonmedical reasons were also obtained from the same area and were randomly selected on the basis of gender and gestational period. Pathological confirmation of the presence/absence of NTDs was completed by experienced pathologists from the Capital Institute of Pediatrics, Beijing, China, in accordance with the International Classification of Diseases, Tenth Revision, codes Q00.0, Q05.9, and Q01.9 (https://www.who.int/standards/classifications/classification-of-diseases). Routine prenatal health checkups, questionnaires, and fetal autopsy reports were completed for all fetuses. All staff involved in the project were specifically trained for this project [5].
Sample Collection and Transportation
All placental samples used for DNA extraction were stored at -20°C in local hospitals before shipment on ice to the study laboratories. Placental tissues were obtained from test and control subjects as previously reported [14].
DNA Extraction
Genomic DNA was extracted from frozen placental tissues using a Blood and Tissue DNA Kit (QIAGEN, Hilden, Germany) in accordance with the manufacturer’s instructions. DNA purity was assessed by measuring light absorbance at 260 nm and 280 nm. Samples with an A260/280 ratio outside of 1.80–1.95 were not used. DNA concentrations were assessed by measuring the absorbance at 260 nm.
SNP Assays for DIO1, DIO2, and DIO3
Nine maternal SNPs were analyzed. We analyzed rs11206237 (A/C) and rs2235544 (A/C), which are located in DIO1; rs12885300 (T/C), rs1388378 (A/C), rs225010 (T/C), rs225011 (C/T), rs225012 (A/G), and rs225014 (T/C), which are located in DIO2; and rs17716499 (C/T), which is located in DIO3. iPLEX Gold SNP genotyping analysis was performed to determine the SNPs in DIO1, DIO2, and DIO3, and the genotypes using the Sequenom MassARRAY System (CapitalBio, Beijing, China) as previously reported [5]. This system uses allele-specific extension in combination with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. PCR and extension primers were designed using RealSNP (https://www.mysequenom.com/), which specifies three primers for each SNP in the assay as follows: two primers for PCR amplification and one primer for an extension reaction via hybridization adjacent to the SNP site (Table 1). SNP assays were successfully performed, and the numbers of pregnant women are shown in Table 2. Unfortunately, we ran out of placental DNA samples from some pregnant women.
Primers for SNP genotyping on DIO1, DIO2, and DIO3 genes
Gene . | Location . | SNPs . | Amplification primers . | Extension primers . |
---|---|---|---|---|
DIO1 | Upstream | rs11206237 | ACGTTGGATGACTTTCTGCTTCGAGGCTTGACGTTGGATGTTCTGGGATGCACCTGTCTG | CTG?TGT?GCT?GAT?TGG?CTC?CAG?CAA?G |
DIO1 | Intronic region | rs2235544 | ACGTTGGATGACCTCTTGCAACTAACCTCCACGTTGGATGCCCTGCAAGAGAAACGAATC | TCAGGCATTCCCAACTT |
DIO2 | Intron | rs225010 | ACGTTGGATGAACCCTGACCTGCAATGAACACGTTGGATGGGCCCCAGGATTCATATTTC | AAC?ATA?ATC?ATA?TTT?GGG?TGA |
DIO2 | Intron | rs225011 | ACGTTGGATGGGTCAGGGTTATCATTAAGTGACGTTGGATGATTAGGGTTAGACTGGGAAG | CGA?ATG?AAT?GCT?AGT?TGT?TAT?AAT?C |
DIO2 | Intron | rs225012 | ACGTTGGATGCCCCACAATTTGTTAACAAGCACGTTGGATGGCAAAGGGAGCACATGAAAC | ACA?TGA?AAC?AAT?TTT?TAT?CTC?TAC |
DIO2 | Nonsynonymous | rs225014 | ACGTTGGATGTCTTCTCCTGGGTACCATTGACGTTGGATGATTCCAGTGTGGTGCATGTC | CTT?TTG?GTG?CAT?GTC?TCC?AGT |
DIO2 | 5'UTR | rs12885300 | ACGTTGGATGTTCATTTCCAAGCACCTATGACGTTGGATGCAAGAAAGAAACAGGCTACG | GGG?AGC?ATA?GAG?ACA?ATG?AAA?G |
DIO2 | Intron | rs1388378 | ACGTTGGATGTCCAGAGACGTTGTAAGAGGACGTTGGATGGGGTAGCCTCAAAATAATGC | GGC?ATC?ACA?CTA?TTT?CAT?AA |
DIO3 | Downstream | rs17716499 | ACGTTGGATGCAGATGGTTTAGCCTTGAGCACGTTGGATGAGCCACCTGTGCCTTCGAG | TAG?AGT?TCA?TAG?AAA?GGG?TCT |
Gene . | Location . | SNPs . | Amplification primers . | Extension primers . |
---|---|---|---|---|
DIO1 | Upstream | rs11206237 | ACGTTGGATGACTTTCTGCTTCGAGGCTTGACGTTGGATGTTCTGGGATGCACCTGTCTG | CTG?TGT?GCT?GAT?TGG?CTC?CAG?CAA?G |
DIO1 | Intronic region | rs2235544 | ACGTTGGATGACCTCTTGCAACTAACCTCCACGTTGGATGCCCTGCAAGAGAAACGAATC | TCAGGCATTCCCAACTT |
DIO2 | Intron | rs225010 | ACGTTGGATGAACCCTGACCTGCAATGAACACGTTGGATGGGCCCCAGGATTCATATTTC | AAC?ATA?ATC?ATA?TTT?GGG?TGA |
DIO2 | Intron | rs225011 | ACGTTGGATGGGTCAGGGTTATCATTAAGTGACGTTGGATGATTAGGGTTAGACTGGGAAG | CGA?ATG?AAT?GCT?AGT?TGT?TAT?AAT?C |
DIO2 | Intron | rs225012 | ACGTTGGATGCCCCACAATTTGTTAACAAGCACGTTGGATGGCAAAGGGAGCACATGAAAC | ACA?TGA?AAC?AAT?TTT?TAT?CTC?TAC |
DIO2 | Nonsynonymous | rs225014 | ACGTTGGATGTCTTCTCCTGGGTACCATTGACGTTGGATGATTCCAGTGTGGTGCATGTC | CTT?TTG?GTG?CAT?GTC?TCC?AGT |
DIO2 | 5'UTR | rs12885300 | ACGTTGGATGTTCATTTCCAAGCACCTATGACGTTGGATGCAAGAAAGAAACAGGCTACG | GGG?AGC?ATA?GAG?ACA?ATG?AAA?G |
DIO2 | Intron | rs1388378 | ACGTTGGATGTCCAGAGACGTTGTAAGAGGACGTTGGATGGGGTAGCCTCAAAATAATGC | GGC?ATC?ACA?CTA?TTT?CAT?AA |
DIO3 | Downstream | rs17716499 | ACGTTGGATGCAGATGGTTTAGCCTTGAGCACGTTGGATGAGCCACCTGTGCCTTCGAG | TAG?AGT?TCA?TAG?AAA?GGG?TCT |
SNP, single nucleotide polymorphism; DIO1, iodothyronine deiodinase type 1; DIO2, iodothyronine deiodinase type 2; DIO3, iodothyronine deiodinase type 3; UTR, untranslated region.
Cochran-Armitage trend test and Fisher’s exact test results among placental samples
. | Cases, N, % . | Controls, N, % . | Cochran-Armitage trend test p value*Fisher’s exact test p value (two-tailed tests) . |
---|---|---|---|
SNPs in DIO1 gene | |||
rs11206237 | |||
AA | 0, 0.0 | 1, 3.3 | 0.656* |
AC | 10, 32.3 | 6, 20.0 | |
CC | 21, 67.7 | 23, 76.7 | |
AA and AC | 10, 32.3 | 7, 23.3 | 0.570 |
CC | 21, 67.7 | 23, 76.7 | |
AA | 0, 0.0 | 1, 3.3 | 0.492 |
AC and CC | 31, 100.0 | 29, 96.7 | |
rs2235544 | |||
AA | 8, 25.8 | 6, 20.0 | 0.995* |
AC | 14, 45.2 | 17, 56.7 | |
CC | 9, 29.0 | 7, 23.3 | |
AA and AC | 22, 71.0 | 23, 76.7 | 0.772 |
CC | 9, 29.0 | 7, 23.3 | |
AA | 8, 25.8 | 6, 20.0 | 0.762 |
AC and CC | 23, 74.2 | 24, 80.0 | |
SNPs located in DIO2 gene | |||
rs225010 | |||
CC | 0, 0.0 | 1, 3.3 | 0.553* |
CT | 18, 58.1 | 13, 43.3 | |
TT | 13, 41.9 | 16, 53.3 | |
CC and CT | 18, 58.1 | 14, 46.7 | 0.446 |
TT | 13, 41.9 | 16, 53.3 | |
CC | 0, 0.0 | 1, 3.3 | 0.492 |
CT and TT | 31, 100.0 | 29, 96.6 | |
rs225011 | |||
CC | 13, 41.9 | 16, 55.2 | 0.476* |
CT | 18, 58.1 | 12, 41.4 | |
TT | 0, 0.0 | 1, 3.4 | |
CC and CT | 31, 100.0 | 28, 96.6 | 0.483 |
TT | 0, 0.0 | 1, 3.4 | |
CC | 13, 41.9 | 16, 55.2 | 0.438 |
CT and TT | 18, 58.1 | 13, 44.8 | |
rs225012 | |||
AA | 0, 0.0 | 1, 3.3 | 0.553* |
AG | 18, 58.1 | 13, 43.3 | |
GG | 13, 41.9 | 16, 53.3 | |
AA and AG | 18, 58.1 | 14, 46.7 | 0.446 |
GG | 13, 41.9 | 16, 53.3 | |
AA | 0, 0.0 | 1, 3.3 | 0.492 |
AG and GG | 31, 100.0 | 29, 96.6 | |
rs225014 | |||
CC | 2, 6.5 | 5, 17.2 | 0.312* |
CT | 20, 64.5 | 17, 58.6 | |
TT | 9, 29.0 | 7, 24.1 | |
CC and CT | 22, 71.0 | 22, 75.9 | 0.774 |
TT | 9, 29.0 | 7, 24.1 | |
CC | 2, 6.5 | 5, 17.2 | 0.247 |
CT and TT | 29, 93.5 | 24, 82.8 | |
rs12885300 | |||
CC | 20, 64.5 | 20, 66.7 | 0.939* |
CT | 10, 32.3 | 10, 33.3 | |
TT | 1, 3.2 | 0, 0.0 | |
CC and CT | 30, 96.8 | 30, 100.0 | 1.000 |
TT | 1, 3.2 | 0, 0.0 | |
CC | 20, 64.5 | 20, 66.7 | 1.000 |
CT and TT | 11, 35.5 | 10, 33.3 | |
rs1388378 | |||
AA | 3, 9.7 | 3, 10.0 | 0.939* |
AC | 13, 41.9 | 12, 40.0 | |
CC | 15, 48.4 | 15, 50.0 | |
AA and AC | 16, 51.6 | 15, 50.0 | 1.000 |
CC | 15, 48.4 | 15, 50.0 | |
AA | 3, 9.7 | 3, 10.0 | 1.000 |
AC and CC | 28, 90.3 | 27, 90.0 | |
SNPs located in DIO3 gene | |||
rs17716499 | |||
CC | 4, 12.9 | 4, 13.3 | 0.943* |
CT | 11, 35.5 | 10, 33.3 | |
TT | 16, 51.6 | 16, 53.3 | |
CC and CT | 15, 48.4 | 14, 46.7 | 1.000 |
TT | 16, 51.6 | 16, 53.3 | |
CC | 4, 12.9 | 4, 13.3 | 1.000 |
CT and TT | 27, 87.1 | 26, 86.7 |
. | Cases, N, % . | Controls, N, % . | Cochran-Armitage trend test p value*Fisher’s exact test p value (two-tailed tests) . |
---|---|---|---|
SNPs in DIO1 gene | |||
rs11206237 | |||
AA | 0, 0.0 | 1, 3.3 | 0.656* |
AC | 10, 32.3 | 6, 20.0 | |
CC | 21, 67.7 | 23, 76.7 | |
AA and AC | 10, 32.3 | 7, 23.3 | 0.570 |
CC | 21, 67.7 | 23, 76.7 | |
AA | 0, 0.0 | 1, 3.3 | 0.492 |
AC and CC | 31, 100.0 | 29, 96.7 | |
rs2235544 | |||
AA | 8, 25.8 | 6, 20.0 | 0.995* |
AC | 14, 45.2 | 17, 56.7 | |
CC | 9, 29.0 | 7, 23.3 | |
AA and AC | 22, 71.0 | 23, 76.7 | 0.772 |
CC | 9, 29.0 | 7, 23.3 | |
AA | 8, 25.8 | 6, 20.0 | 0.762 |
AC and CC | 23, 74.2 | 24, 80.0 | |
SNPs located in DIO2 gene | |||
rs225010 | |||
CC | 0, 0.0 | 1, 3.3 | 0.553* |
CT | 18, 58.1 | 13, 43.3 | |
TT | 13, 41.9 | 16, 53.3 | |
CC and CT | 18, 58.1 | 14, 46.7 | 0.446 |
TT | 13, 41.9 | 16, 53.3 | |
CC | 0, 0.0 | 1, 3.3 | 0.492 |
CT and TT | 31, 100.0 | 29, 96.6 | |
rs225011 | |||
CC | 13, 41.9 | 16, 55.2 | 0.476* |
CT | 18, 58.1 | 12, 41.4 | |
TT | 0, 0.0 | 1, 3.4 | |
CC and CT | 31, 100.0 | 28, 96.6 | 0.483 |
TT | 0, 0.0 | 1, 3.4 | |
CC | 13, 41.9 | 16, 55.2 | 0.438 |
CT and TT | 18, 58.1 | 13, 44.8 | |
rs225012 | |||
AA | 0, 0.0 | 1, 3.3 | 0.553* |
AG | 18, 58.1 | 13, 43.3 | |
GG | 13, 41.9 | 16, 53.3 | |
AA and AG | 18, 58.1 | 14, 46.7 | 0.446 |
GG | 13, 41.9 | 16, 53.3 | |
AA | 0, 0.0 | 1, 3.3 | 0.492 |
AG and GG | 31, 100.0 | 29, 96.6 | |
rs225014 | |||
CC | 2, 6.5 | 5, 17.2 | 0.312* |
CT | 20, 64.5 | 17, 58.6 | |
TT | 9, 29.0 | 7, 24.1 | |
CC and CT | 22, 71.0 | 22, 75.9 | 0.774 |
TT | 9, 29.0 | 7, 24.1 | |
CC | 2, 6.5 | 5, 17.2 | 0.247 |
CT and TT | 29, 93.5 | 24, 82.8 | |
rs12885300 | |||
CC | 20, 64.5 | 20, 66.7 | 0.939* |
CT | 10, 32.3 | 10, 33.3 | |
TT | 1, 3.2 | 0, 0.0 | |
CC and CT | 30, 96.8 | 30, 100.0 | 1.000 |
TT | 1, 3.2 | 0, 0.0 | |
CC | 20, 64.5 | 20, 66.7 | 1.000 |
CT and TT | 11, 35.5 | 10, 33.3 | |
rs1388378 | |||
AA | 3, 9.7 | 3, 10.0 | 0.939* |
AC | 13, 41.9 | 12, 40.0 | |
CC | 15, 48.4 | 15, 50.0 | |
AA and AC | 16, 51.6 | 15, 50.0 | 1.000 |
CC | 15, 48.4 | 15, 50.0 | |
AA | 3, 9.7 | 3, 10.0 | 1.000 |
AC and CC | 28, 90.3 | 27, 90.0 | |
SNPs located in DIO3 gene | |||
rs17716499 | |||
CC | 4, 12.9 | 4, 13.3 | 0.943* |
CT | 11, 35.5 | 10, 33.3 | |
TT | 16, 51.6 | 16, 53.3 | |
CC and CT | 15, 48.4 | 14, 46.7 | 1.000 |
TT | 16, 51.6 | 16, 53.3 | |
CC | 4, 12.9 | 4, 13.3 | 1.000 |
CT and TT | 27, 87.1 | 26, 86.7 |
SNP, single nucleotide polymorphism; DIO1, iodothyronine deiodinase type 1; DIO2, iodothyronine deiodinase type 2; DIO3, iodothyronine deiodinase type 3; N, number.
Associations between SNPs and NTDs were determined by means of two different tests (the Cochran-Armitage trend test and the Fisher’s exact test). Individual SNP of DIOs has three genotypes; for example, rs1120637 in the gene DIO1 has genotypes AA, AC, and CC. However, we do not know which allele is dominant. In order to investigate whether the SNPs of DIOs are associated with NTDs, at first, we conducted the Cochran-Armitage trend test for three categories; then we allocated three genotypes into two categories and ran the Fisher’s exact test for two categories. The way to classify each category is to add two of them together and leave the remaining one as another (for example, [i] AA and AC, CC; [ii] AA, AC and CC of rs1120637 in the gene DIO1). Fisher’s exact test for the 2 × 2 contingency table and Cochran-Armitage trend test for the 3 × 2 contingency table were performed.
Differences were considered statistically significant at p < 0.05. p value obtained by the Cochran-Armitage test was marked with an asterisk (*), and the Fisher’s exact test is presented without any marks.
Statistical Analysis
Associations between SNPs and NTDs were determined by the Cochran-Armitage trend test and Fisher’s exact test using JMP version 15 (SAS Institute Inc., Cary, NC, USA). Differences were considered statistically significant at p < 0.05.
This case-control-based study has only two phenotypes, “control and NTD fetuses.” Associations between SNPs and NTDs were determined by means of two different tests (the Cochran-Armitage trend test and the Fisher’s exact test). Individual SNPs of DIOs have three genotypes, for example, rs1120637 in the gene DIO1 has genotypes AA, AC, and CC. However, we do not know which allele is dominant. In order to investigate whether the SNPs of DIOs are associated with NTDs, at first, we conducted the Cochran-Armitage trend test for three categories; then we allocated three genotypes into two categories and ran the Fisher’s exact test for two categories. The way to classify each category is to add two of them together and leave the remaining one as another (for example, (i) AA and AC, CC; (ii) AA, AC and CC of rs1120637 in the gene DIO1). Fisher’s exact test for the 2 × 2 contingency table and the Cochran-Armitage trend test for the 3 × 2 contingency table were performed (Table 2).
Ethics Approval and Consent to Participate
The study protocol was reviewed and approved by the Ethics Boards of the Capital Institute of Pediatrics, Beijing, China (cited March 8, 2004); Teikyo University, Japan (Tei-I-Rin 14-010); and Osaka Medical College, Japan (2758-01). All participants provided written informed consent.
Results
In this study, we analyzed DIO1, DIO2, and DIO3 from frozen placental samples using SNP assays. In our previous study, we found that pregnant women with high levels of serum homocysteine and low thyroid hormone levels were associated with NTDs. In this study, we analyzed nine SNPs in DIO1, DIO2, and DIO3 using placental samples. However, no statistically significant association was found between the SNPs and NTDs through the Cochran-Armitage test for trends or the Fisher’s exact test (p > 0.05) (Table 2).
Discussion
In this study, we found no statistically significant association between placental SNPs of DIO1, DIO2, and DIO3 and NTDs. Moreover, although our previous report showed a relationship between NTDs and pregnant women with high serum homocysteine levels and low thyroid hormone levels, the maternal SNPs of the DIO genes are not statistically related to NTDs in this population [5]. Therefore, other factors, such as thyroid hormone receptors and transporters in the placenta which are involved in the passage of maternal thyroid hormone to fetuses should be investigated in pregnant women in this area.
The fetal development depends on maternal thyroid hormones, particularly before the onset of endogenous fetal thyroid hormone production at approximately 14–16 weeks of gestation [7]. The placenta is known to be a rich source of DIOs [8, 9]. DIO1 catalyzes the monodeiodination of T4 to T3. DIO2 is the primary activating enzyme that locally catalyzes the monodeiodination of T4 to T3. The highest activities of DIO2 have been reported in the central nervous system. Locally generated T3 in the fetal brain converted from maternally transported T4 has been reported to be essential for normal early brain development [6, 10]. DIO3 is the deactivating enzyme that catalyzes the monodeiodination of T4 to reverse T3 and of T3 to T2. DIO3 is primarily present in the placenta and, to a lesser extent, in the central nervous system [6, 11]. Despite the relatively high DIO3 activity in the placenta, physiologically important levels of maternal T4 are transferred to the fetus [15, 16]. DIO3 plays an essential role in regulating thyroid hormone inactivation during embryonic development. Altered levels of transplacental thyroid hormone during passage have an effect on neurological development.
DIO1, DIO2, and DIO3 are seleno-enzymes that affect thyroid hormone regulation in a wide range of tissues including the placenta. Deficiency of selenium, iodine, folic acid, and other vitamins among women living in the area of Lüliang, China, where our study was previously reported [1‒3]. Moreover, in areas of high prevalence of NTDs, coexistence of high maternal level of tHCY and low levels of thyroid hormone increased the risk of NTDs in a case-control study performed previously [4, 5]. However, the question remains as to why fetuses in some pregnant women in the control group who had coexistence of high levels of maternal tHCY and low levels of thyroid hormone did not develop as NTDs in their fetuses. Therefore, we aimed to explore metabolism of thyroid hormone in the placenta of those pregnant women.
So far, there are a few reports about SNPs of DIOs in placental samples or in samples from pregnant women. It was reported that a SNP in DIO2 (rs225014) of the placenta is associated with disrupted placental activity but not with dysglycemia or adverse gestational outcomes among consecutive singleton-pregnant Brazilian pregnant women aged 18–45 years old [17]. Moreover, SNPs in DIO2 (rs225010, rs225012, and rs1388378) were reported to be associated with child mental retardation in the iodine-deficient mountainous areas, Qinba region of China, which were located nearby the area of our study and both areas had similar geography and economic situations [12, 13]. It was also reported that rs11206244 of DIO1 of Romanian pregnant women was associated with pregnancy outcome of preeclampsia delivered neonates with very low birth weight at a significantly low gestational age [18]. A study performed among Spanish pregnant women showed evidence of epidemiological effect modification of both DIO1 (rs2235544-CC) and DIO2 (rs12885300-CC and -CT) that resulted in altered strength of negative associations between prenatal phenol exposure and thyroid hormone [19], suggesting DIO1 and DIO2 among pregnant women are more sensitive to certain environmental factors. Therefore, we selected 9 SNPs of DIOs (as shown in Tables 1,,2). We selected rs11206237 in DIO1 rather than rs1120644 because rs1120644 was reported to be associated with non-NTD outcomes among pregnant women [18]. Considering that the metabolism of pregnant women is not the same as that of a non-pregnant adult, rs225011 in DIO2 and rs17716499 in DIO3, that had been reported few times, were also included.
Most studies on SNPs were performed by means of statistical analysis. However, potential associations between deiodinase polymorphism and risk of pregnancy outcomes are noteworthy, as described above. In this study, no statistical association was found between polymorphisms of DIOs and NTDs among pregnant women. Other factors, such as placental thyroid hormone transporters, receptors, and epigenetic modification of methylation and homocysteinylation in those pregnant women, also may affect passage of maternal thyroid hormone to fetuses. It was reported that in mouse, porcine, and cattle models, the effect of DIO3 variants on DIO3 activity might be influenced by the epigenetic process of genomic imprinting. Effects of DIO3 polymorphisms on thyroid hormone homeostasis depend on the parental origin of the variant allele [20‒22]. Therefore, we aim to study epigenetic modification of methylation and homocysteinylation including genomic imprinting in our future research.
To our knowledge, the effect of DIO3 variants on functional characterization of the DIO3 protein has not been studied. Peeters et al. [23] have reported that using mouse model in the central nervous system, upregulation of DIO2 and downregulation of DIO3 are physiological components in response to iodine deficiency. Pregnant women in this study are living in areas of iodine deficiency; whether this evidence may occur in the human placenta or not, further confirmation is required.
As previously reported, low levels of maternal thyroid hormones are involved in folic acid metabolism by influencing maternal riboflavin metabolism, oxidative stress, and the vitamin D biosynthetic pathway [5] (shown in Fig. 1[24‒27]). Therefore, abnormal levels of maternal thyroid hormones may alter the fetal metabolism of folic acid. Moreover, our previous case study [28] showed that (1) in human fetuses with spina bifida, there is possible abnormal crosstalk between thyroid hormones and retinoic acid signaling through their common retinoid X receptors and the subsequent recruitment of histone modifications; and (2) human fetuses with spina bifida in the context of maternal serum hyperthyroidism present elevated thyroid hormone signaling along with T3 degradation (attenuated DIO2/DIO3 switching). Therefore, abnormal levels of maternal thyroid hormones may be involved in fetal metabolism of folic acid by influencing various maternal and fetal metabolic and biosynthetic pathways, rather than SNPs of placental DIOs (shown in Fig. 1).
Placental SNPs of DIO1, DIO2, and DIO3 that were investigated in this study and the maternal SNPs of DIO1, DIO2, and DIO3 from our previous study [5] are not associated with NTDs, despite lower maternal serum T4 levels augmenting the risk of higher maternal serum tHCY levels, as previously reported [5]. Our results suggest that although maternal thyroid hormones influence the balance between one-carbon metabolism and other metabolic signaling pathways [5, 24, 25, 26, 27] involved in NTDs, the placental SNPs of DIO1, DIO2, and DIO3 are not associated with NTDs. Vit. B2, vitamin B2; Vit. B6, vitamin B6; Vit. B12, vitamin B12; Vit. D, vitamin D; FMN, flavin mononucleotide; FAD, flavin adenine dinucleotide; MTHFR, methylenetetrahydrofolate reductase; CH2-THF, 5,10-methylenetetrahydrofolate; CH3-THF, 5-methyl tetrahydrofolate; SAM, S-adenosylmethionine; Met, methionine; MTRR, methionine synthase reductase; MTR, methionine synthase; Hcy, homocysteine; CBS, cystathionine ß-synthase; Se, selenium; T3, tri-iodothyronine; T4, thyroxine; SNP, single nucleotide polymorphism; tHCY, total homocysteine; DIO1, iodothyronine deiodinase type 1; DIO2, iodothyronine deiodinase type 2; DIO3, iodothyronine deiodinase type 3; NTD, neural tube defect.
Placental SNPs of DIO1, DIO2, and DIO3 that were investigated in this study and the maternal SNPs of DIO1, DIO2, and DIO3 from our previous study [5] are not associated with NTDs, despite lower maternal serum T4 levels augmenting the risk of higher maternal serum tHCY levels, as previously reported [5]. Our results suggest that although maternal thyroid hormones influence the balance between one-carbon metabolism and other metabolic signaling pathways [5, 24, 25, 26, 27] involved in NTDs, the placental SNPs of DIO1, DIO2, and DIO3 are not associated with NTDs. Vit. B2, vitamin B2; Vit. B6, vitamin B6; Vit. B12, vitamin B12; Vit. D, vitamin D; FMN, flavin mononucleotide; FAD, flavin adenine dinucleotide; MTHFR, methylenetetrahydrofolate reductase; CH2-THF, 5,10-methylenetetrahydrofolate; CH3-THF, 5-methyl tetrahydrofolate; SAM, S-adenosylmethionine; Met, methionine; MTRR, methionine synthase reductase; MTR, methionine synthase; Hcy, homocysteine; CBS, cystathionine ß-synthase; Se, selenium; T3, tri-iodothyronine; T4, thyroxine; SNP, single nucleotide polymorphism; tHCY, total homocysteine; DIO1, iodothyronine deiodinase type 1; DIO2, iodothyronine deiodinase type 2; DIO3, iodothyronine deiodinase type 3; NTD, neural tube defect.
A limitation of this study was the small number of subjects because (1) NTDs are a rare disease and (2) the location of this study was completed in a rural area that is not conveniently located for sample storage and transportation. There was no refrigerated cargo transportation at that time, and the frozen samples were transported to places by car with plenty of ice. Our future research would aim to focus on functional studies in placenta samples with larger sample sizes.
Acknowledgments
The authors are grateful to all participating hospitals, medical staff, subjects of this study, and their families for their assistance with the collection of samples and clinical information.
Statement of Ethics
The study protocol was reviewed and approved by the Ethics Boards of the Capital Institute of Pediatrics, Beijing, China (cited March 8, 2004); Teikyo University, Japan (Tei-I-Rin 14-010); and Osaka Medical College, Japan (2758-01). All participants provided written informed consent.
Conflict of Interest Statement
The authors confirm that there are no conflicts of interest.
Funding Sources
The authors disclose receipt of the following financial support for the research, authorship, and/or publication of this article: this research has been supported by the Public Service Development and Reform Pilot Project of the Beijing Medical Research Institute (BMR2019-11), Grant-in-Aid for Scientific Research of Japan (grant number KAKENHI 15K08823), and a grant from the Japan China Medical Association 2010.
Author Contributions
F.W. carried out the field research, questionnaire, and samples transport and wrote original draft preparation; Y.-H.G. did hypothesis, design, and formal analysis and wrote original draft preparation; J.G., Y.B. and Z.Q. participated in field research, informed consent, data collection, and/or laboratory tests; P.Z. cleaned data and did formal analysis; M.U. and M.M. did formal statistical analysis; T.Z. performed supervision, project administration, and funding acquisition; all authors read and approved the final manuscript.
Additional Information
Fang Wang and Yan-Hong Gu contributed equally to this work.
Data Availability Statement
All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author. A preprint version of this article is available on Research Square [DOI: https://doi.org/10.21203/rs.3.rs-1511857/v1].