[Genetic analysis of a fetus with Meckel syndrome due to variants of TMEM67 gene].

OBJECTIVE: To carry out prenatal diagnosis for a fetus with Meckel syndrome (MKS) and explore its genetic basis. METHODS: A pregnant woman presented at Suzhou Municipal Hospital in February 2018 was selected as the study subject. Clinical data was collected. Muscle tissue sample from the abortus and peripheral blood samples from the couple were collected. Genomic DNA was extracted and subjected to chromosomal microarray analysis (CMA) and whole exome sequencing. Candidate variant was verified by Sanger sequencing. RESULTS: The fetus was found to have microcephaly, oligohydramnios, polycystic kidneys and banana-shaped cerebellum at 18 weeks of gestation. After induction of labor, it was found to have encephalocele, renal cysts and polydactyly. CMA has found no abnormality. Whole exome sequencing revealed novel compound heterozygous variants c.296delA (p.Lys99SerfsTer6) and c.1243G>A (p.Val415Met) in the TMEM67 gene. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the c.296delA variant was predicted to be pathogenic (PVS1+PM2_Supporting+PP4), whilst the c.1243G>A variant was predicted to be likely pathogenic (PM2_Supporting+PM3+PP3_Moderate+PP4). CONCLUSION: The c.296delA and c.1243G>A compound heterozygous variants of the TMEM67 gene probably underlay the MKS in this fetus.

LncRNA DANCR restrained the survival of mycobacterium tuberculosis H37Ra by sponging miR-1301-3p/miR-5194.

Tuberculosis is a worldwide contagion caused by Mycobacterium tuberculosis (MTB). MTB is characterized by intracellular parasitism and is semi-dormant inside host cells. The persistent inflammation caused by MTB can form a granuloma in lesion regions and intensify the latency of bacteria. In recent years, several studies have proven that long non-coding RNAs (lncRNAs) play critical roles in modulating autophagy. In our study, the Gene Expression Omnibus (GEO) databases were searched for lncRNAs that are associated with tuberculosis. We found that lncRNA differentiation antagonizing non-protein coding RNA (DANCR) increased in the peripheral blood samples collected from 54 pulmonary tuberculosis patients compared to 23 healthy donors. By constructing DANCR overexpression cells, we analyzed the possible cellular function of DANCR. After analyzing our experiments, it was found that the data revealed that upregulation of DANCR facilitated the expression of signal transducer and activator of transcription 3, autophagy-related 4D cysteine peptides, autophagy-related 5, Ras homolog enriched in the brain, and microtubule-associated protein 1A/1B light chain 3 (STAT3, ATG4D, ATG5, RHEB, and LC3, respectively) by sponging miR-1301-3p and miR-5194. Immunofluorescence analysis indicated that DANCR played a positive role in both autophagosome formation and fusion of autolysosomes in macrophages. The colony-forming unit (CFU) assay data also showed that the cells overexpressing DANCR were more efficient in eliminating the intracellular H37Ra strain. Consequently, these data suggest that DANCR restrained intracellular survival of M. tuberculosis by promoting autophagy via miR-1301-3p and miR-5194.

Neonatal cytokines associated with infant overweight and obesity at 1 year of age.

OBJECTIVE: The incidence of childhood overweight and obesity has been increasing in recent years. Immune dysregulation has been demonstrated as a condition related to childhood obesity. Whether the neonatal immune status is related to infant overweight and obesity at 1 year of age is unclear. METHODS: To explore the relationship between neonatal cytokines and infant overweight and obesity, we conducted a prospective study in Suzhou Municipal Hospital Affiliated to Nanjing Medical University from 2015 to 2016. 514 neonates were recruited and their dried blood spots were collected after birth. Infants were grouped into normal size groups and overweight and obesity groups based on BMI at 1 year of age. 27 neonatal cytokines levels were compared between the two groups. RESULTS: 370 infants were included in final analysis. Granulocyte colony stimulating factor (GCSF), interleukin-17A (IL17A) and platelet derived growth factor-BB (PDGF-BB) levels were independently associated with childhood overweight and obesity (OR =1.27, 95%CI 1.03, 1.57; OR =1.29, 95%CI: 1.06, 1.60; OR =0.69, 95%CI: 0.49, 0.96). Additionally, neonatal GCSF and IL17A levels were positively associated with increased BMI (ß = 0.11, 95%CI: 0.02, 0.19; ß = 0.07, 95%CI 0.01, 013) and BMI z-scores (ß = 0.10, 95%CI: 0.02, 0.18; ß = 0.06, 95%CI 0.01, 0.13). Neonatal PDGF-BB levels were negatively associated with BMI (ß = -0.12, 95%CI: -0.23, -0.01) and BMI z-scores (ß = -0.12, 95%CI: -0.23, -0.01). The inverse probability weighting (IPW) was performed to account for potential selection bias of this study, and the results were consistent with the above mentioned findings. CONCLUSIONS: Neonatal GCSF, IL17A and PDGF-BB levels were correlated with infant overweight and obesity at 1 year of age, suggesting that early life immune status play a significant role of late obesity.

Gut Microbiota Exceeds Cervical Microbiota for Early Diagnosis of Endometriosis.

The diagnosis of endometriosis is typically delayed by years for the unexclusive symptom and the traumatic diagnostic method. Several studies have demonstrated that gut microbiota and cervical mucus potentially can be used as auxiliary diagnostic biomarkers. However, none of the previous studies has compared the robustness of endometriosis classifiers based on microbiota of different body sites or demonstrated the correlation among microbiota of gut, cervical mucus, and peritoneal fluid of endometriosis, searching for alternative diagnostic approaches. Herein, we enrolled 41 women (control, n = 20; endometriosis, n = 21) and collected 122 well-matched samples, derived from feces, cervical mucus, and peritoneal fluid, to explore the nature of microbiome of endometriosis patients. Our results indicated that microbial composition is remarkably distinguished between three body sites, with 19 overlapped taxa. Moreover, endometriosis patients harbor distinct microbial communities versus control group especially in feces and peritoneal fluid, with increased abundance of pathogens in peritoneal fluid and depletion of protective microbes in feces. Particularly, genera of Ruminococcus and Pseudomonas were identified as potential biomarkers in gut and peritoneal fluid, respectively. Furthermore, novel endometriosis classifiers were constructed based on taxa selected by a robust machine learning method. These results demonstrated that gut microbiota exceeds cervical microbiota in diagnosing endometriosis. Collectively, this study reveals important insights into the microbial profiling in different body sites of endometriosis, which warrant future exploration into the role of microbiota in endometriosis and highlighted values on gut microbiota in early diagnosis of endometriosis.

Whole-exome sequencing identified a novel mutation in CHM of a Chinese family.

Choroideraemia (CHM) is a rare X-linked progressive-inherited retinal disease. In this study, we diagnosed and explored the genetic cause in a Chinese pedigree exhibiting nyctalopia and decreased visual acuity in early life. Clinical data and peripheral blood samples were collected from available family members. Sanger sequencing of RPGR and RP2 genes, and subsequently whole-exome sequencing was carried out to investigate the molecular cause. The proband was initially diagnosed as retinitis pigmentosa and experienced night blindness at an early age and decreased visual acuity in teens. The other affected males in this family suffered from the same problem. Direct sequencing failed to reveal the genetic cause and hence a novel hemizygous mutation c.861_862insGCTT was detected by WES in CHM gene resulting in a premature stop codon and a truncated protein. Subsequently, it was confirmed by Sanger sequencing and cosegregation analysis. We describe a novel mutation c.861_862insGCTT in CHM gene in a Chinese pedigree with choroideraemia. Our study emphasizes the utilization of next-generation sequencing in the diagnosis and genetic analysis of retinal diseases.

Association of three missense mutations in the homocysteine-related MTHFR and MTRR gene with risk of polycystic ovary syndrome in Southern Chinese women.

BACKGROUND: The etiology between homocysteine and polycystic ovary syndrome (PCOS) is unclear. In humans, the level of homocysteine is mainly affected by two enzymes: methylene tetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR). While the activity of these two enzymes is mainly affected by three missense mutations, namely C677T (MTHFR), A1298C (MTHFR), and A66G (MTRR). This study aims to examine the association between the three missense mutations and PCOS and investigate whether the three missense mutations exerted their effect on PCOS by affecting the homocysteine level. METHODS: A case-control study was designed, comprising 150 people with PCOS and 300 controls. Logistic regression analysis was used to assess the association between the three missense mutations and PCOS. Linear regression analysis was used to assess the association between the three missense mutations and the homocysteine level. Mediation analysis was used to investigate whether the three missense mutations exerted their effect on PCOS by affecting the homocysteine level. RESULTS: Following adjustments and multiple rounds of testing, MTHFR A1298C was found to be significantly associated with PCOS in a dose-dependent manner (compared to AA, OR = 2.142 for AC & OR = 3.755 for CC; P < 0.001). MTRR A66G was nominally associated with PCOS. Mutations in MTHFR A1298C and MTRR A66G were significantly associated with the homocysteine level. Mediation analysis suggested the effect of MTHFR A1298C on PCOS was mediated by homocysteine. CONCLUSIONS: MTHFR A1298C and MTRR A66G were associated with PCOS, and MTHFR A1298C might affect the risk of PCOS by influencing the homocysteine level.

Clinical Utility of SNP Array Analysis in Prenatal Diagnosis: A Cohort Study of 5000 Pregnancies.

BACKGROUND: Single nucleotide polymorphism array (SNP-array) has been introduced for prenatal diagnosis. We aimed to evaluate the clinical value of SNP-array in the diagnosis of fetal chromosomal anomalies. METHODS: A retrospective study was conducted on 5000 cases tested by SNP-array, and the results of 4022 cases analyzed by both karyotyping and SNP-array were compared. RESULTS: SNP-array analysis of 5000 samples revealed that the overall abnormality detection rate by SNP-array was 12.3%, and the overall detection rate of clinically significant copy number variations (CNVs) by SNP-array was 2.6%. SNP-array identified clinically significant submicroscopic CNVs in 4.5% fetuses with anomaly on ultrasonography, in 1.6% of fetuses with advanced maternal age (AMA), in 2.5% of fetuses with abnormal result on maternal serum screening, in 2.9% of fetuses with abnormal non-invasive prenatal testing (NIPT) results and in 3.0% of fetuses with other indications. Of the 4022 samples analyzed by both karyotyping and SNP-array, SNP-array could identify all the aneuploidy and triploidy detected by karyotyping but did not identify balanced structural chromosomal abnormalities and low-level mosaicism detected by karyotyping. CONCLUSION: SNP-array could additionally identify clinically significant submicroscopic CNVs, and we recommend the combination of SNP-array analysis and karyotyping in prenatal diagnosis.

Correction to: miR-205-5p inhibits human endometriosis progression by targeting ANGPT2 in endometrial stromal cells.

An amendment to this paper has been published and can be accessed via the original article.

[Prenatal diagnosis of a fetus with cleft lip and palate by using chromosomal microarray analysis].

OBJECTIVE: To explore the genetic basis for a fetus with cleft lip and palate. METHODS: Copy number variations (CNVs) in the fetus and his parents were detected with chromosomal microarray analysis (CMA). RESULTS: As revealed by the CMA assay, the fetus has carried a 228 kb deletion in Xp11.22 region and a 721 kb duplication in 9p21.1. Both CNVs were inherited from the parents. The CNV in Xp11.22 was predicted to be pathogenic by involving the PHF8 gene, whilst the CNV in 9p21.1 was predicted to be benign. CONCLUSION: Deletion of the Xp11.22 region probably underlies the cleft lip and palate in this fetus.

Development of a highly thermostable immunoassay based on a nanobody-alkaline phosphatase fusion protein for carcinoembryonic antigen detection.

Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM-5) assays are employed in routine clinical settings to diagnose tumor. We selected two nanobodies with high-affinity to CEACAM-5, termed Nb11C12 and Nb2D5, using phage-display technology. The Nb2D5 fused with calf intestinal alkaline phosphatase (CAP), human placental alkaline phosphatase (HAP), or Pyrococcus abyssi alkaline phosphatase (PAP) were expressed in human embryonic kidney (HEK293) cells. The enzymatic activity of Nb2D5-HAP fusion protein was the best and remained stable at 60 °C for 7 days. The affinity of Nb2D5-HAP fusion protein to CEACAM-5 reached 42 pM. A chemiluminescent enzyme immunoassay (CLEIA) based on Nb2D5-HAP fusion protein was established for quantitative CEACAM-5 assay in clinical settings. The CLEIA exhibited a wide linear range of 0.31-640 ng/mL toward CEACAM-5, with a limit of detection (LOD) of 0.85 ng/mL. No cross-reactivity occurred with CEACAM-1, CEACAM-3, CEACAM-6, or CEACAM-8, and no interference was observed with rheumatoid factors. The CLEIA based on Nb2D5-HAP fusion protein was stable for 8 weeks at 37 °C and 50% relative humidity. The CLEIA developed from Nb2D5-HAP fusion protein had much better stability and linearity with similar reproducibility compared with the enzyme-linked immunosorbent assay developed from conventional monoclonal antibodies, which have been widely used in clinics over the past several decades. Graphical abstract.

Two heterozygous mutations in the ERCC6 gene associated with Cockayne syndrome in a Chinese patient.

OBJECTIVE: To confirm diagnosis and explore the genetic aetiology in a Chinese patient suspected to have Cockayne syndrome (CS). METHODS: The patient was clinically examined, and the patient and her biological parents underwent genetic analysis using whole exome sequencing (WES) and Sanger sequencing. The foetus of the patient's mother underwent prenatal diagnostic Sanger sequencing using amniotic fluid obtained at 19 weeks' gestation. RESULTS: Clinical examination of the patient showed developmental delay, progressive neurologic dysfunction and premature aging. Two compound, heterozygous ERCC excision repair 6, chromatin remodelling factor (ERCC6) gene mutations were detected in the proband by WES and confirmed by Sanger sequencing, comprising a known paternal nonsense mutation (c.643G > T, p.E215X) and a novel maternal short insertion and deletion mutation (c.1614_c.1616delGACinsAAACGTCTT, p.K538_T539delinsKNVF). The patient was consequently diagnosed with CS type I. The foetus of the patient's mother was found to carry only the maternally-derived c.1614_c.1616delGACinsAAACGTCTT variant. CONCLUSION: This study emphasized the value of WES in clinical diagnosis, and enriched the known spectrum of ERCC6 gene mutations.

Experimental factors are associated with fetal fraction in size selection noninvasive prenatal testing.

Every year, 4-6 million pregnant women undergo noninvasive prenatal testing (NIPT), which is used worldwide for fetal aneuploidy screening. Adequate fetal cell-free DNA (cfDNA) is the critically important factor to ensure high sensitivity and specificity. In this study, we sought to increase the fetal fraction by adjusting experimental factors in the size selection for NIPT. CfDNA was extracted from 1495 pregnant women at 12-26 weeks of gestation for sequencing of shorter cfDNA NIPT (< 140 bp). Multivariable linear regression models were used to evaluate the association between experimental factors and fetal fraction. Nomograms for the likelihood of high fetal fraction (> 20%) were constructed according to significant factors in multivariable regression models. Our results suggested that cfDNA and library concentrations were negatively correlated with fetal fraction, and uniquely mapped reads were positively correlated with fetal fraction. Lower cfDNA and library concentrations, shorter cfDNA fragments, and higher uniquely mapped reads may be more conducive to obtaining higher fetal fractions. Furthermore, we constructed easy-to-use nomograms incorporating the maternal, fetal characteristics and experimental factors to precisely predict the probability of high fetal fraction with an area under the curve (AUC) of 0.773 (95% confidence interval: 0.749-0.797). Collectively, our maternal plasma cfDNA-based nomograms consider experimental factors that can be adjusted and may improve a laboratory's ability to obtain higher fetal cfDNA concentrations.

miR-205-5p inhibits human endometriosis progression by targeting ANGPT2 in endometrial stromal cells.

BACKGROUND: miRNA expression profiles in ectopic endometrium (EC) serving as pathophysiologic genetic fingerprints contribute to determining endometriosis progression; however, the underlying molecular mechanisms remain unknown. METHODS: miRNA microarray analysis was used to determine the expression profiling of EC fresh tissues. qRT-PCR was performed to screen miR-205-5p expression in EC tissues. The roles of miR-205-5p and its candidate target gene, angiopoietin-2 (ANGPT2), in endometriosis progression were confirmed on the basis of both in vitro and in vivo systems. miR-205-5p and ANGPT2 expression were measured by in situ hybridization and immunochemistry, and their clinical significance was statistically analysed. RESULTS: miR-205-5p was screened as a novel suppressor of endometriosis through primary ectopic endometrial stromal cell migration, invasion, and apoptosis assay in vitro, along with endometrial-like xenograft growth and apoptosis in vivo. In addition, ANGPT2 was identified as a direct target of miR-205-5p through bioinformatic target prediction and luciferase reporter assay. Re-expression and knockdown of ANGPT2 could respectively rescue and simulate the effects induced by miR-205-5p. Importantly, the miR-205-5p-ANGPT2 axis was found to activate the ERK/AKT pathway in endometriosis. Finally, miR-205-5p and ANGPT2 expression were closely correlated with the endometriosis severity. CONCLUSION: The newly identified miR-205-5p-ANGPT2-AKT/ERK axis illustrates the molecular mechanism of endometriosis progression and may represent a novel diagnostic biomarker and therapeutic target for disease treatment.

The long intergenic non-protein coding RNA 707 promotes proliferation and metastasis of gastric cancer by interacting with mRNA stabilizing protein HuR.

Multiple studies have revealed that long non-coding RNAs (lncRNAs) extensively participate in human cancer malignant progression. The long intergenic non-protein coding RNA 707 (LINC00707), 3087 bp in length, was recently reported to be an essential oncogene in promoting lung adenocarcinoma cell proliferation and metastasis. However, its role in gastric cancer (GC) remains unclear. In this study, we identified that LINC00707 was excessively expressed in GC tissues and correlated with advanced stage, larger tumor size, lymph node metastasis and poorer prognosis in GC patients. In vitro and in vivo assays showed that LINC00707 promote GC cell proliferation and metastasis. Mechanistically, LINC00707 could abundantly interact with mRNA stabilizing protein HuR; "LINC00707-HuR" coalition ulteriorly combined with VAV3/F11R mRNAs and increased their stability. Taken together, our findings prove that LINC00707 may act as an oncogene in GC by regulating mRNA stability and serve as a potential target for GC diagnosis and prognosis.

Submicroscopic chromosomal imbalances contribute to early abortion.

BACKGROUND: Chromosomal abnormalities are one of the genetic mechanisms associated with abortion. However, the roles of submicroscopic chromosomal imbalances in early abortion are still unclear. This study aims to find out whether submicroscopic chromosomal imbalances contribute to early abortion. METHODS: A total of 78 chorionic villus specimens from early spontaneous abortion patients with no obvious abnormality are collected after miccroassay analysis (the case group). At the same time, 60 chorionic villus specimens from induced abortion patients with no obvious abnormality are selected as the control group. The submicroscopic structures of chromosomes from two groups are analyzed using an array-based comparative genomic hybridization (aCGH). RESULTS: In the case group, 15 specimens show submicroscopic chromosomal abnormalities including 14 micro-deletion/micro-duplication in chromosomes 2, 4, 5, 6, 7, 8, 9, 12, 15, 16, 18, and 22, and 1 uniparental disomy (UPD) in chromosome 19. Moreover, no pathogenic copy number variations are found in the control group. The results between these two groups exhibit significantly statistical difference. CONCLUSION: Submicroscopic chromosomal imbalances may be one of the main reasons for early abortion.

Whole exome sequencing identifies a pathogenic mutation in WFS1 in two large Chinese families with autosomal dominant all-frequency hearing loss and prenatal counseling.

OBJECTIVES: To identify the pathogenic mutation and provide prenatal counseling and diagnosis in two large Chinese families with autosomal dominant all-frequency hearing loss. METHODS: Whole exome sequencing technology was used to identify the pathogenic mutation of the two families. In addition, 298 patients with sporadic hearing loss and 400 normal controls were studied to verify the mutation/polymorphism nature of the identified variant. Prenatal diagnosis was carried out. RESULTS: A rare missense mutation c.2389G > A (p.D572N) in the Wolframin syndrome 1 (WFS1) gene was identified. It was reported in only one previous Chinese study, and never in other populations/ethnicities. The mutation was also found in one patient with sporadic hearing loss (1/298, 0.3%). A healthy baby was born after prenatal diagnosis. CONCLUSION: Our findings strongly suggest that the c.2389G > A mutation in WFS1 is associated with all-frequency hearing loss, rather than low- or high-frequency loss. So far, the mutation is only reported in Chinese. Prenatal diagnosis and prenatal counseling is available for these two Chinese families.

Identification of a novel nonsense mutation of the neurotrophic tyrosine kinase receptor type 1 gene in two siblings with congenital insensitivity to pain with anhidrosis.

Objective To explore the aetiology of congenital insensitivity to pain with anhidrosis (CIPA) in two Chinese siblings with typical CIPA symptoms including insensitivity to pain, inability to sweat, and self-mutilating behaviours. Methods Clinical examination and genetic testing were conducted of all available family members, and the findings were used to create a pedigree. Mutation screening using PCR amplification and DNA Sanger sequencing of the entire neurotrophic tyrosine kinase receptor type 1 gene ( NTRK1) including intron-exon boundaries was used to identify mutations associated with CIPA. Results A novel nonsense mutation (c.7C > T, p. Arg3Ter) and a known splice-site mutation (c.851-33 T > A) were detected in NTRK1 and shown to be associated with CIPA. Conclusion Our findings expand the known mutation spectrum of NTRK1 and provide insights into the aetiology of CIPA.

Combining the Advantages of Alkene and Azo E-Z Photoisomerizations: Mechanistic Insights into Ketoimine Photoswitches.

We carried out CASPT2//(TD)DFT and CASPT2//CASSCF studies on the working mechanism of imine switches, including a camphorquinone-derived ketoimine (shortened as k-Imine) switch designed by Lehn as well as a model camphorquinone alkene-imine (a-Imine) proposed in this study. Under the experimental conditions (light irradiation with 455 and 365 nm for E and Z, respectively), k-Imine is excited to the S1:(nN,π*) state and then decays toward a perpendicular intermediate following the C═N bond rotation coordinate. During the bond rotation, a mild energy barrier caused by the strong interaction of S1:(nN,π*) and S2:(nO,π*) states will more or less slow down the rotation speed of k-Imine. The large difference in irradiation light wavelength supports k-Imine as a two-way photoswitch. The photoisomerization of a-Imine obeys a similar but fully barrierless pattern while requiring a higher excitation energy to reach the (nN,π*) state. The good directionality of thermal isomerization toward E(a-Imine), plus the barrierless photoisomerization, allows for the design of a thermal and photo-operated switch. For both imines, a minimal-energy crossing point (MECI) located at the perpendicular region, with low relative energy and close to the rotary path, ensures the directionality of C═N bond rotation and confirms imines as optimal candidates for photoswitches and motors.

Detection of complex deletions in chromosomes 13 and 21 in a fetus by noninvasive prenatal testing.

BACKGROUND: To detect complex fetal subchromosomal abnormalities by noninvasive prenatal testing (NIPT). CASE PRESENTATION: After routine prenatal serum screening, the plasma of high-risk pregnant women were tested via NIPT, and the NIPT results were further validated by fetal karyotype analysis and array-based comparative genomic hybridization (aCGH) through amniocentesis. In addition, the chromosome karyotypes of the parents were also analyzed. NIPT results indicated subchromosomal abnormalities in chromosomes 13 and 21; aCGH results showed 22 Mb and 16 Mb deletions in 13 q31.3 - q34 and 21q11.1 - q21.3, respectively; and the fetal karyotype was 45,XX, der(13),-21. The maternal karyotype 46,XX,inv(9)(p12q13),t(13;21)(q31.3;q21.3) was abnormal, while the paternal karyotype showed no obvious abnormality. CONCLUSION: In this study, we successfully detected complex deletions in chromosomes 13 and 21 in a fetus using NIPT, and NIPT can provide effective genetic information for the detection of fetal subchromosomal abnormalities.

Methylenetetrahydrofolate reductase polymorphisms at 3'-untranslated region are associated with susceptibility to preterm birth.

BACKGROUND: Etiology and mechanism of preterm birth (PTB) is complicated. Genetic susceptibility is one of the key factors involved in the pathogenic mechanism underlying PTB. METHODS: A subset of single nucleotide polymorphisms (SNPs) selected by bioinformatics approach from 3'-untranslated region (3'-UTR) of methylenetetrahydrofolate reductase (MTHFR) gene were subjected to SNaPshot analysis in a case-control study. Three SNPs (rs45451599, rs1537515, rs1537516) were simultaneously tested in one tube, among 1,135 DNA samples including 480 PTBs and 655 term controls. RESULTS: Two perfectly correlated (r(2)=1) SNPs, rs1537515 and rs1537516, were found significantly associated with PTB susceptibility [P=0.012; OR: 0.65; 95% confidence interval (CI), 0.47-0.91]. The frequencies of the minor alleles were lower in PTB cases than in controls, which the frequencies were 0.066 in PTB cases and 0.095 in controls. G and T allele frequencies of rs1537515 were the same with rs1537516 (P=0.011; OR: 0.666; 95% CI, 0.49-0.91). Rs45451599 was not found associated with PTB (P=0.52; OR: 0.76; 95% CI, 0.33-1.74). The 18-25 nucleotides in length of microRNAs (miRNAs) which can regulate gene expressions are involved in binding partial complementary sequences within 3'-UTR. The two loci are at 3'-UTR of MTHFR mRNA. Rs1537516 is a potential target of miR-1304-3p, while rs1537515 is miR-1224-3p and miR-3150-5p. CONCLUSIONS: In conclusion, rs1537515 and rs1537516 within the 3'-UTR of the MTHFR gene may be associated with susceptibility to PTB.