Comparison of single nucleotide polymorphisms in the 3′ untranslated region of HLA-G in placentas between spontaneous preterm birth and preeclampsia
Lee et al. BMC Res Notes
Comparison of single nucleotide polymorphisms in the 3′ untranslated region of HLA-G in placentas between spontaneous preterm birth and preeclampsia
Ji Young Lee 0
Hyun Mi Kim 0
Mi Ju Kim 0
Hyun‑Hwa Cha 0
Won Joon Seong 0
0 Department of Obstetrics and Gynecology, Kyungpook National University Hospital, Kyungpook National University School of Medicine , 130 Dongdeok‐ro, Jung‐gu, Daegu 700‐721 , South Korea
Objective: To compare single nucleotide polymorphisms (SNPs) in the 3′‑ untranslated region (3′UTR) of human leukocyte antigen (HLA)‑ G in placentas between spontaneous preterm birth and preeclampsia pregnancies. Results: Placental samples matched for gestational age were obtained from 20 cases of spontaneous preterm births and 19 cases of preeclampsia. Genomic deoxyribonucleic acid was extracted from placenta tissue and the 3′UTR region of HLA‑ G was amplified via polymerase chain reaction. Nine SNPs were analyzed by direct Sanger sequencing. There was no significant difference in gestational age at delivery or birth weight between two groups. And there were no significant differences in the allele and phenotype frequencies between two groups.
HLA‑ G; Single nucleotide polymorphism; Placenta; Preeclampsia; Preterm births
Preterm births and preeclampsia (PE) are the major
complications that contribute to morbidities during
pregnancy; however, its pathophysiology has not been clearly
identified. Immunological maladaptation in the
maternal–fetal interface has accounted for the pathogenesis of
adverse pregnancy outcomes, including PE, intrauterine
growth restrictions, spontaneous preterm births, and
congenital infections [
]. The human leukocyte antigen
(HLA) system is a major histocompatibility complex
(MHC) protein in humans. Among the HLA antigens,
the human leukocyte antigen-G (HLA-G) antigen is
expressed only in humans and has a proposed role in
protecting the extravillous trophoblast from the
maternal immune system [
]. In addition, several
researchers have reported that reduced expression of HLA-G
is linked to PE [
], and the HLA-G protein has been
reported to be linked to spontaneous preterm birth and
intra-amniotic inflammation or infections [
nucleotide polymorphisms (SNPs) are the most common
type of genetic variations and are defined by at least two
variants, one of which is present at a frequency greater
than 1% [
]. Recently, associations between
preeclampsia and SNPs in the 3′ untranslated region (3′UTR)
of HLA-G have been observed . However, these
studies only compared placentas from cases of preeclampsia
and normal full term pregnancies without consideration
of gestational age at delivery. Therefore, the aim of the
present study was to compare SNPs in the 3′UTR of the
HLA-G between pregnancies complicated with
spontaneous preterm births and those with preeclampsia in
gestational age matched placentas.
We collected placental samples from 20 cases with
spontaneous preterm births and 19 cases of preeclampsia.
The placental samples 1 cm× 1 cm in size were
aseptically obtained from the fetal side and kept in a frozen
state (− 70 °C). In all cases of preeclampsia in this study,
the patients had blood pressures ≥ 140/90 mmHg,
dipstick protein ≥ 1+/4+, or 24-h urine protein ≥ 300 mg/
dl. For gestational age matching, cases of spontaneous
preterm birth were selected with a similar gestational age
at delivery for each case of preeclampsia. The indications
for delivery in preterm birth group included preterm
labor and preterm premature rupture of the membranes
without other maternal or fetal indications. All
pregnant women enrolled in this study gave written informed
consent before participation. This study was approved
by the Institutional Review Board (IRB) of Kyungpook
National University, Daegu, South Korea (IRB File No:
Deoxyribo nucleic acid (DNA) extraction
Genomic DNA was extracted from placenta tissue, using
the Qiagen QIAamp Fast DNA tissue Kit (Qiagen,
Valencia, CA, USA). DNA concentration was determined
using a NanoDrop ND-1000 spectrophotometer(Thermo
Fisher Scientific, Wilmington, DE) and purity was
assessed based on the 260/280 nm absorbance ratio from
1.7 to 2.1.
Polymerase chain reaction (PCR) and PCR product purification
Nine SNPs in the 3′UTR of HLA-G that were previously
] were evaluated in this study as follows;
rs371194629, rs1707, rs1710, rs17179101, rs17179108,
rs1063320, rs9380142, rs1610696, rs1233331. The DNA
sequences including the 3′UTR region of HLA-G were
amplified via PCR in a final reaction volume of 50 μl,
using the Multiplex PCR Master Mix (Qiagen, Valencia,
CA, USA) and 10 pmol of each primeralong with 100 ng
of genomic DNA using SimpliAmp™ Thermal Cycler(Life
Technologies, Carlsbad, CA, USA). Size of PCR amplicon
was 526 bp. The PCR parameters were as follows: initial
denaturation of 95 °C for 15 min, followed by 35 cycles
of 94 °C for 30 s, 60 °C for 90 s, 72 °C for 40 s, and a final
extension of 72 °C for 10 min. Amplified PCR products
were purified using the GeneAll expin Kit (GeneALL
Biotechnology, Seoul, Korea.).
Genotyping by Sanger sequencing
Sanger sequencing was performed using the BigDye
Terminator V 3.1 Cycle Sequencing Kit (Applied Biosystems,
USA). Sequencing results were compared with reference
ENST00000428701.5), using the alignment program
BLAST V2.5.0 of NCBI. The positions of nine SNP
were manually compared using CodonCode Aligner
V.5.1.5(CodonCode Corporation, Centerville, MA, USA).
The Hardy–Weinberg (HW) equilibrium and Chi square
test were used to verify the significance of differences in
the genotypic data and allelic frequencies between two
groups. Chi square test was two-sided test and
statistical significance was defined when p value was lower than
0.05. The Haploview software (V 4.2) was employed to
perform the linkage disequilibrium (LD) and haplotype
analysis among nine of the genotyped SNPs. LD patterns
were constructed using an algorithm designed by Gabriel
et al. [
] with a minor allel frequency (MAF) of ≥ 1%.
The clinical characteristics of women with
spontaneous preterm subjects and PE are listed in Table 1. There
were no significant differences between the two groups
in terms of gestational age at delivery and birth weight
(32.8 ± 4.8 vs. 33.3 ± 4.4, p = 0.74, 2.19 ± 0.9 vs. 1.80 ± 0.8,
p = 0.17). Haploview showed the genotype distributions
were in HW equilibrium except rs17179101 in
pregnancies without preeclampsia. The allele frequencies of all
the SNPs did not show any statistical differences between
the two groups (Table 2). Furthermore, there were no
statistical differences between the control group and cases
of PE in terms of genotype frequencies for all nine SNPs
In this study, we compared nine SNPs in the 3′UTR
region of HLA-G between spontaneous preterm birth
and preeclampsia in gestational age matched placentas.
And we found that there were no differences in allelic
frequencies and genotypic distributions between the
two groups. HLA-G has been reported to contribute to
maternal immune tolerance at the maternal–fetal
interface through natural killer cell receptors by blocking their
cytotoxic effect [
]. Previously, SNPs in the 3′UTR region
of HLA-G have been evaluated in terms of preeclampsia
T test and Chi square test, both two-sided, was used to analyze the data and
statistical significance was defined when p value was lower than 0.05
a Mean ± standard deviation; GAD, gestational age at delivery
Chi square test, both two-sided, was used to analyze the data and statistical
significance was defined when p value was lower than 0.05
pathophysiology, which showed diverse results [
Among the nine variations, no statistically significant
differences have been shown in genotype distributions for
rs1610696, rs1707, rs1710, rs17179101, rs17179108, and
rs1233331 between the control subjects and cases of PE
. In contrast, the distribution of the A or G allele of
rs9380142 and the C or G allele of rs1063320 was found
to be different between the control subjects and cases of
]. In particular, rs1704 has been analyzed in
several studies [
], which showed the presence
of a 14-bp insertion that was significantly higher in cases
of PE [
16, 19, 21
]. Recently, Quach et al. suggested that a
pair of alleles in the 3′UTR of HLA-G (rs17179101 C/C
allele and rs938021 G/G) may play a role in the
pathophysiology of PE [
Furthermore, HLA-G has been investigated with
regards to preterm birth or intraamniotic inflammations/
infections. Soluble HLA-G (sHLA-G) has been detected
in both the maternal serum/plasma and amniotic fluid,
and its concentrations are influenced by the gestational
age and several maternal or fetal complications,
including fetal gender, fetal neural tube defect, intraamniotic
inflammations/infections, and preeclampsia [
7, 9, 22–24
Kusanovic et al. investigated sHLA-G in amniotic fluid
and found that it was elevated in preterm parturition and
associated with intraamniotic inflammation/infection [
Additionally, higher vaginal and maternal serum
concentration of sHLA-G in cases of preterm premature rupture
of membranes may be associated with local or systemic
]. Interestingly, the concentration of
maternally circulating sHLA-G are significantly lower
in patients with PE even before its manifestation [
]. Likewise, it is well known that HLA-G is involved in
the pathophysiology of spontaneous preterm labor and
PE; however, studies on HLA-G in spontaneous preterm
birth have mainly investigated sHLA-G only, not the
3′UTR in HLA-G. Therefore, our study was meaningful
because we analyzed and compared SNPs in the 3′UTR
of HLA-G between placentas with spontaneous
preterm birth and those with preeclampsia. In addition, we
matched the gestational age at delivery, and the results
showed similar birth weights between the two groups.
Previous studies only compared HLA-G in the placentas
from cases of PE versus normal term pregnancy and PE.
In conclusion, we found that there were no significant
differences in the SNPs in the 3′UTR of HLA-G between
spontaneous preterm birth and PE. Further studies with a
larger cohort are warranted.
The presented data is limited by sample size. For
gestational age matching, we did not compare
preeclampsia with normal pregnancy, but compared spontaneous
preterm birth with preeclampsia due to gestational age
HLA‑ G: human leukocyte antigen‑ G; SNP: single nucleotide polymorphisms;
3′UTR: 3′‑untranslated region; PCR: polymerase chain reaction; PE: preeclamp ‑
sia; MHC: major histocompatibility complex.
CHH, LJY, KHM, KMJ, and SWJ participated in the patient’s enrollment. CHH
was responsible for the design of the study, the acquisition, analysis, and inter‑
pretation of data; and drafting and revising the study. JYL was responsible for
the drafting and revising the manuscript. WJS contributed to study concept
and design. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
All data generated or analyzed in this study are included in this published.
Consent for publication
Ethical approval and consent to participate
This study was approved by the Institutional Review Board (IRB) of Kyung‑
pook National University, Daegu, South Korea (IRB File No: 2016‑07‑005). We
obtained written informed consent from all participants. The biospecimens for
this study were provided by National Biobank of Korea‑Kyungpook National
University Hospital (KNUH), which is supported by the Ministry of Health, Wel‑
fare and affairs. All materials derived from the National Biobank of Korea‑KNUH
were obtained (with informed consent) under institutional review board
This research was supported by Kyungpook National University research fund,
Springer Nature remains neutral with regard to jurisdictional claims in pub‑
lished maps and institutional affiliations.
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