Error analysis of stages involved in CBCT-guided implant placement with surgical guides when different printing technologies are used.

STATEMENT OF PROBLEM: Digital light processing (DLP), continuous liquid interface printing (CLIP), and stereolithography (SLA) technologies enable 3-dimensional (3D) printing of surgical guides. However, how their accuracy compares and how accuracy may affect subsequent steps in guided surgery is unclear. PURPOSE: The purpose of this in vitro study was to investigate the fabrication and seating accuracy of surgical guides printed by using DLP, SLA, and CLIP technologies and evaluate the positional deviation of the osteotomy site and placed implant compared with the digital implant plan. MATERIAL AND METHODS: Twenty-one polyurethane models were divided into 3 groups and used to plan implants and design surgical guides. The guides were fabricated by using DLP, SLA, or CLIP 3D printers (n=7) and scanned, and the scan file was compared with the digital design file to analyze the fabrication accuracy at the intaglio and overall external surfaces using root mean square (RMS) values. The triple scan protocol was used to evaluate the seating accuracy of the guides on their respective models. Osteotomies were prepared on models by using the guides followed by a microcomputed tomography image of each osteotomy. The implants were placed through the guides, the scan bodies were tightened to implants, and the models were scanned to obtain the images of placed implant position. Osteotomy and placed implant images were used to calculate the entry point, apex, and long axis deviations from the planned implant position with a software program. A 2-way repeated-measures ANOVA of the RMS data was used to analyze printing and seating trueness, and homogeneity of variance analyses were used at each surface for precision. A 3-way repeated-measures ANOVA was used to analyze distance deviations over the stages (osteotomy and final implant) and locations studied, and a 2-way repeated-measures ANOVA was used for angular deviations. Homogeneity of variance analyses were performed for precision (α=.05). RESULTS: The 3D printer type significantly affected the trueness of the guide at the intaglio surface (P<.001). SLA guides had the lowest mean RMS (59.04 µm) for intaglio surface, while CLIP had the highest mean RMS (117.14 µm). Guides from all 3D printers had low variability among measured deviations and therefore were similarly precise. The seating accuracy of SLA and DLP guides was not significantly different, but both had lower mean RMS values than CLIP (P=.003 for SLA, P=.014 for DLP). There were no significant interactions between the stage of surgery, the printer type, or the location of implant deviation (P=.734). Only the location of deviation (cervical versus apical) had a significant effect on distance deviations (P<.001). The printer type, stage of surgery, and their interaction did not significantly affect angular deviations (P=.41). CONCLUSIONS: The 3D printing technology affected printing trueness. The intaglio surface trueness was higher with SLA and overall trueness was higher with the CLIP printer. The precision of all guides was similarly high. Guides from SLA and DLP printers had more accurate seating than those from CLIP. Higher deviations were observed at the apex; however, osteotomy and final implant position did not significantly differ from the digitally planned position.

Fast Focal Point Correction in Prism-Coupled Total Internal Reflection Scanning Imager Using an Electronically Tunable Lens.

Total internal reflection (TIR) is useful for interrogating physical and chemical processes that occur at the interface between two transparent media. Yet prism-coupled TIR imaging microscopes suffer from limited sensing areas due to the fact that the interface (the object plane) is not perpendicular to the optical axis of the microscope. In this paper, we show that an electrically tunable lens can be used to rapidly and reproducibly correct the focal length of an oblique-incidence scanning microscope (OI-RD) in a prism-coupled TIR geometry. We demonstrate the performance of such a correction by acquiring an image of a protein microarray over a scan area of 4 cm² with an effective resolution of less than 20 microns. The electronic focal length tuning eliminates the mechanical movement of the illumination lens in the scanning microscope and in turn the noise and background drift associated with the motion.

Characterization of protein expression levels with label-free detected reverse phase protein arrays.

In reverse-phase protein arrays (RPPA), one immobilizes complex samples (e.g., cellular lysate, tissue lysate or serum etc.) on solid supports and performs parallel reactions of antibodies with immobilized protein targets from the complex samples. In this work, we describe a label-free detection of RPPA that enables quantification of RPPA data and thus facilitates comparison of studies performed on different samples and on different solid supports. We applied this detection platform to characterization of phosphoserine aminotransferase (PSAT) expression levels in Acanthamoeba lysates treated with artemether and the results were confirmed by Western blot studies.

Calibration of oblique-incidence reflectivity difference for label-free detection of a molecular layer.

Oblique-incidence reflectivity difference (OI-RD) is a form of polarization-modulation ellipsometry that measures properties of thin films on a solid surface through the change in polarization state of light upon reflection from the surface. The measurement accuracy depends on the precision of the phase modulation amplitude and azimuthal alignments of key polarizing optical elements and, thus, requires careful calibration. In the present work, we describe robust methods of such calibrations that enable precise determination of the modulation amplitude and static retardation of a phase modulator and azimuths of key polarizing optics in an OI-RD system.

Mutational Analysis of TCOF1, GSC, and HOXA2 in Patients With Treacher Collins Syndrome.

Treacher Collins syndrome is an autosomal dominant craniofacial malformation mainly caused by mutations in the TCOF1 gene. Few cases have been observed in the Chinese population. Herein, the authors report the mutational analysis of TCOF1, GSC, and HOXA2 to determine the mutational features of the 3 genes in Chinese patients with Treacher Collins syndrome. Genomic DNA of the patients and their parents was extracted from peripheral blood following a standard protocol. DNA sequencing analysis was performed on all exons and the exon-intron borders of TCOF1, GSC, and HOXA2 in addition to the 1200-bp upstream of TCOF1. Four novel single nucleotide polymorphisms were detected in TCOF1, one of which was in the promoter region. Mutations in GSC and HOXA2 were not found in the 3 patients. Our results suggest the possibility of genetic heterogeneity or different mechanisms leading to the disease. Further functional study of the alteration is necessary to obtain more definitive information.

Developing an Efficient and General Strategy for Immobilization of Small Molecules onto Microarrays Using Isocyanate Chemistry.

Small-molecule microarray (SMM) is an effective platform for identifying lead compounds from large collections of small molecules in drug discovery, and efficient immobilization of molecular compounds is a pre-requisite for the success of such a platform. On an isocyanate functionalized surface, we studied the dependence of immobilization efficiency on chemical residues on molecular compounds, terminal residues on isocyanate functionalized surface, lengths of spacer molecules, and post-printing treatment conditions, and we identified a set of optimized conditions that enable us to immobilize small molecules with significantly improved efficiencies, particularly for those molecules with carboxylic acid residues that are known to have low isocyanate reactivity. We fabricated microarrays of 3375 bioactive compounds on isocyanate functionalized glass slides under these optimized conditions and confirmed that immobilization percentage is over 73%.

Tissue factor pathway inhibitor-2 is critical in zebrafish cardiogenesis.

Human tissue factor pathway inhibitor-2 (Tfpi-2) is an extracellular matrix-associated Kunitz-type serine proteinase inhibitor and plays an important role in various cellular processes. We have previously shown that zebrafish Tfpi-2 (zTfpi-2) mainly expressed in the brain and heart of zebrafish, and it is involved in the development of central nervous system. Here, we identified zTfpi-2 as an evolutionarily conserved protein essential for zebrafish heart development, as embryos depleted of zTfpi-2 failed to undergo cardiogenesis. Changes of cardiogenic markers, vmhc, amhc and bmp4, confirmed zTfpi-2 knockdown caused cardiac defects, including retrenched ventricle, enlarged atrium and malformation of atrioventricular boundary. The sarcomeric organization was also disrupted by embryonic depletion of zTfpi-2, thus establishing the functional role of zTfpi-2 in cardiac contractility. In addition, hematopoietic defects were detected in the zTfpi-2-deficiency embryos. Importantly, injection of ztfpi-2 mRNA attenuated those cardiac and hematopoietic defects. Taken together, this study demonstrated a critical role of zTfpi-2 during embryonic cardiac development, as well as an important regulator of hematopoiesis.

Effect of Paricalcitol Combined with Cinacalcet on Calcium and Phosphorus Metabolism in Patients Receiving Maintenance Hemodialysis.

This study was designed to compare the beneficial effects of paricalcitol combined with or without cinacalcet on calcium and phosphorus metabolism in patients undergoing maintenance hemodialysis (MHD). A total of 140 patients who received MHD in our hospital from March 2021 to March 2022 were randomly divided into a control group (intravenous paricalcitol, n = 70) and a test group (intravenous paricalcitol combined with oral cinacalcet, n = 70). Clinical baseline data and relevant laboratory parameters before treatment were compared. Additionally, calcium, phosphorus, intact parathyroid hormone in serum were measured and compared between the 2 groups before treatment and 1, 2, 3, 4, 5, 6, 9, 10, and 12 months after treatment. As a result, comparison before treatment demonstrated no significant difference in baseline data such as age, sex, and most laboratory parameters between the 2 groups (P > .05), but there was a significant difference in mean corpuscular volume (P < .001). The serum phosphorus level decreased and calcium level increased significantly in the 2 groups after treatment, while the intact parathyroid hormone level showed no significant change within 12 months of treatment (P > .05). In addition, the combined treatment for 6-12 months caused a much lower phosphorus level (P < .05) and higher calcium level (P < .05) than the treatment with paricalcitol alone, and the difference increased with the extension of treatment time. Collectively, paricalcitol combined with cinacalcet, which is more effective than paricalcitol alone, has a positive effect on calcium and phosphorus metabolism in patients receiving MHD.

Low expression of TFPI-2 associated with poor survival outcome in patients with breast cancer.

BACKGROUND: The purpose of this study is to evaluate the prognostic value of TFPI-2 expression in breast cancer patients through examining the correlation between TFPI-2 expression and breast cancer clinicopathologic features. METHODS: Immunohistochemical staining combined with digital image analysis was used to quantify the expression of TFPI-2 protein in breast tumor tissues. For evaluation of the prognostic value of TFPI-2 expression to each clinicopathologic factor, Kaplan-Meier method and COX's Proportional Hazard Model were employed. RESULTS: TFPI-2 expression was significantly correlated with tumor size, lymph node metastasis, histologic grade, clinical stage, and vessel invasion. More importantly, TFPI-2 expression was also associated with disease-free survival (DFS) of breast cancer patients. We found that patients with high TFPI-2 expression had longer DFS compared with those with low or negative expression of TFPI-2 (P <0.05, log-rank test). Cox's regression analysis indicated that TFPI-2 expression, histologic grade, and vessel invasion might be significant prognostic factors for DFS, while TFPI-2 expression and histologic grade were the most significant independent predictors for tumor recurrence. Compared with the group with low/high TFPI-2 expression, the TFPI-2 negative group was more likely to have tumor relapse. The hazard ratio of DFS is 0.316 (P <0.01). CONCLUSIONS: Low or negative expression of TFPI-2 is associated with breast cancer progression, recurrence and poor survival outcome after breast cancer surgery. TFPI-2 expression in breast tumors is a potential prognostic tool for breast cancer patients.

An audit of autopsy-confirmed diagnostic errors in perinatal deaths: What are the most common major missed diagnoses.

Perinatal autopsies are essential to establish the cause of stillbirth or neonatal death and improve clinical practice. Limited studies have provided detailed major missed diagnoses of perinatal deaths in current clinical practice. In this retrospective audit of 177 perinatal autopsies including 99 stillbirths and 78 neonatal deaths with complete pathologic evaluation, 66 cases (21 Class I and 45 Class II diagnostic errors) were revealed as major discrepancies (37.3%), with complete agreements in 80 cases (45.2%). The difference in major discrepancies between stillbirth and neonatal death groups was significant (P < 0.001), with neonatal deaths being more prone to Class I errors. Various respiratory diseases (25/66, 37.9%) and congenital malformations (16/66, 24.2%) accounted for the majority of missed diagnoses (41/66, 62.1%). More importantly, neonatal respiratory distress syndrome (NRDS) was the most common type I missed diagnosis (7/8, 87.5%), markedly higher than the average 11.9% of all Class I errors. Our findings suggest that there are high disparities between clinical diagnoses and autopsy findings in perinatal deaths, and that various respiratory diseases are mostly inclined to cause major diagnostic errors. We first demonstrated that NRDS is the most common type I missed diagnosis in perinatal deaths, which clinicians should pay special attention to in practice.

WWC1/2 regulate spinogenesis and cognition in mice by stabilizing AMOT.

WWC1 regulates episodic learning and memory, and genetic nucleotide polymorphism of WWC1 is associated with neurodegenerative diseases such as Alzheimer's disease. However, the molecular mechanism through which WWC1 regulates neuronal function has not been fully elucidated. Here, we show that WWC1 and its paralogs (WWC2/3) bind directly to angiomotin (AMOT) family proteins (Motins), and recruit USP9X to deubiquitinate and stabilize Motins. Deletion of WWC genes in different cell types leads to reduced protein levels of Motins. In mice, neuron-specific deletion of Wwc1 and Wwc2 results in reduced expression of Motins and lower density of dendritic spines in the cortex and hippocampus, in association with impaired cognitive functions such as memory and learning. Interestingly, ectopic expression of AMOT partially rescues the neuronal phenotypes associated with Wwc1/2 deletion. Thus, WWC proteins modulate spinogenesis and cognition, at least in part, by regulating the protein stability of Motins.

miRNA-135a promotes breast cancer cell migration and invasion by targeting HOXA10.

BACKGROUND: miRNAs are a group of small RNA molecules regulating target genes by inducing mRNA degradation or translational repression. Aberrant expression of miRNAs correlates with various cancers. Although miR-135a has been implicated in several other cancers, its role in breast cancer is unknown. HOXA10 however, is associated with multiple cancer types and was recently shown to induce p53 expression in breast cancer cells and reduce their invasive ability. Because HOXA10 is a confirmed miR-135a target in more than one tissue, we examined miR-135a levels in relation to breast cancer phenotypes to determine if miR-135a plays role in this cancer type. METHODS: Expression levels of miR-135a in tissues and cells were determined by poly (A)-RT PCR. The effect of miR-135a on proliferation was evaluated by CCK8 assay, cell migration and invasion were evaluated by transwell migration and invasion assays, and target protein expression was determined by western blotting. GFP and luciferase reporter plasmids were constructed to confirm the action of miR-135a on downstream target genes including HOXA10. Results are reported as means ± S.D. and differences were tested for significance using 2-sided Student"s t-test. RESULTS: Here we report that miR-135a was highly expressed in metastatic breast tumors. We found that the expression of miR-135a was required for the migration and invasion of breast cancer cells, but not their proliferation. HOXA10, which encodes a transcription factor required for embryonic development and is a metastasis suppressor in breast cancer, was shown to be a direct target of miR-135a in breast cancer cells. Our analysis showed that miR-135a suppressed the expression of HOXA10 both at the mRNA and protein level, and its ability to promote cellular migration and invasion was partially reversed by overexpression of HOXA10. CONCLUSIONS: In summary, our results indicate that miR-135a is an onco-miRNA that can promote breast cancer cell migration and invasion. HOXA10 is a target gene for miR-135a in breast cancer cells and overexpression of HOXA10 can partially reverse the miR-135a invasive phenotype.

PolyA RT-PCR-based quantification of microRNA by using universal TaqMan probe.

Quantification of microRNAs (miRNAs) in tissues under normal and pathological conditions is important for elucidating miRNA functions. Based on a PolyA RT-PCR method we have described (J Zhang et al. Biochem Biophys Res Commun 2008 377:136-140), a modified miRNA quantification method was developed and validated using a universal TaqMan probe complementary to the reverse transcript primer. This method effectively detects miRNA expression in cell lines and tissues. The TaqMan probe is more accurate and reliable than the SYBR Green method since it was free from primer dimers. A series of miRNAs were tested in five different mouse tissues: the method differentiated different miRNAs of the same family. This universal TaqMan probe-based PolyA RT-PCR method showed its advantages in precision, simplicity and high-throughput capability compared with other miRNA-detecting methods.

ZNF191 alters DNA methylation and activates the PI3K-AKT pathway in hepatoma cells via transcriptional regulation of DNMT1.

BACKGROUND: Alteration of DNA methylation is an important event in pathogenesis and progression of hepatocellular carcinoma (HCC). DNA methyltransferase (DNMT) 1, the foremost contributor in DNA methylation machinery, was revealed elevated in HCC and significantly correlates with poor prognosis. However, the transcriptional regulation of DNMT1 in HCC remains unknown. METHODS: Real-time PCR and immunohistochemistry were performed to detect DNMT1 and zinc finger transcription factor 191 (ZNF191) expressions in HCCs. Transcription activity of DNMT1promoter was analyzed with Luciferase reporter activity assay. The binding capacity of ZNF191 protein to DNMT1 promoter was examined with chromatin immunoprecipitation-qPCR (ChIP-qPCR) and electrophoretic mobility shift assay (EMSA). DNA methylation level of hepatoma cells was detected with Methylation array. RESULTS: ZNF191 can regulate DNMT1 mRNA and protein expression positively, and increase the transcription activity of the DNMT1 promoter. ChIP-qPCR and EMSA revealed that ZNF191 protein directly binds to the DNMT1 promoter at nt-240 AT(TCAT)3 TC. Moreover, DNMT1 and ZNF191 expression correlate positively in human HCCs. With methylation array, DNA methylation alteration was observed in hepatoma cells with ZNF191 knockdown, and the differential methylation sites are enriched in the PI3K-AKT pathway. Furthermore, we proved DNMT1 contributes the effect of ZNF191 on hepatoma cell growth via the PI3K-AKT pathway. CONCLUSION: ZNF191 is a novel transcription regulator for DNMT1, and the pro-proliferation effect of ZNF191/DNMT1/p-AKT axis in hepatoma cells implies that ZNF191 status in HCCs may affect the therapeutic effect of DNMTs inhibitors and PI3K inhibitors for precise treatment of the disease.

Mutational analysis of GSC, HOXA2 and PRKRA in 106 Chinese patients with microtia.

OBJECTIVE: Microtia is defined as a developmental malformation characterized by a small, abnormal shaped auricle, with atresia or stenosis of the auditory canal. Genes responsible for nonsyndromic microtia have remained elusive. We therefore report a mutational analysis of GSC, HOXA2 and PRKRA in 106 congenital microtia patients without any combined malformation to explore the relationship between GSC, HOXA2, PRKRA and nonsyndromic microtia. METHODS: A total of 106 patients with a clinical diagnosis of congenital microtia and a control group (100 unaffected controls) were recruited through the Eye and ENT Hospital of Fudan University in China. Genomic DNA was extracted following a standard protocol. DNA sequencing analysis was performed in all exons and the exon-intron borders of GSC, HOXA2 and PRKRA. RESULTS: We identified 5 genomic variants in GSC, HOXA2 and PRKRA. As to the GSC, we obtained a reported variant g.994C > T in exon 2, which resulted in no change of protein. Our results revealed that g.994C > T was also detected in 10 control cases. We also detected 2 novel variants, g.90G > A and g.114A > C, in the 5'UTR of HOXA2. No class 5 or 4 genomic variant of PRKRA was identified in our microtia patients. Additionally, two previously reported SNVs in GSC and PRKRA were also presented. CONCLUSIONS: We suggest that g.994C > T is a new SNV, which is different from the previous report. Further study is needed to prove the function of 2 novel variants in the 5'UTR of HOXA2, and to explore the possible mechanism of these variants in the occurrence of microtia.

Comprehensive expression profiling of microRNAs in laryngeal squamous cell carcinoma.

BACKGROUND: MicroRNAs (miRNAs) are noncoding RNAs involved in posttranscriptional regulation of gene expression in cancer and provide new perspectives on the development of laryngeal squamous cell carcinoma (SCC). METHODS: miRNA expression of 6 pairs of laryngeal SCC and adjacent normal tissues was screened using miRNA array. Laser capture microdissection was applied to isolate a homogeneous group of cells from laryngeal SCC samples. The results of miRNA array analysis were validated in 48 pairs of laryngeal SCC and adjacent normal tissues using quantitative RT-PCR. RESULTS: Twenty-nine differentially expressed miRNAs were detected in the 6 pairs of laryngeal SCC, of which 6 were confirmed, including upregulation of miR-21, miR-93, miR-205, and miR-708 and downregulation of miR-125b and miR-145. Their putative target genes were predicted using 3 online software programs. CONCLUSION: These differentially expressed miRNAs may play a role in tumorigenesis and progression in laryngeal SCC and offer new angles for further investigations into the function of miRNAs.

MicroRNA expression profiling and target genes study in congenital microtia.

OBJECTIVE: Microtia is a complicated congenital anomaly with a genetic and environmental predisposition, and the molecular events underlying this disease are not fully understood. MicroRNAs (miRNAs) are a class of 20-22 nucleotide non-coding RNAs that function to control post-transcriptional gene expression. We want to find the miRNA expression profiling of microtia by using Affymetrix GeneChip(®) miRNA 2.0 Arrays. METHODS: We selected 9 microtia cartilages and 3 normal controls for GeneChip(®) miRNA 2.0 Arrays analysis. The altered miRNA were analyzed by poly (A) RT-PCR from 58 microtia samples and 16 normal controls. We predicted the target genes of miRNAs by bioinformatics and RT-PCT was used to confirm the target genes. RESULTS: We found 11 miRNAs with significantly altered expression in the microtic group compared to the normal controls, which included 6 up-regulated miRNAs and 5 down-regulated miRNAs. These miRNAs were further examined using poly (A) RT-PCR analysis, we found that miR-451 and miR-486-5p were significantly up-regulated and miR-200c was significantly down-regulated in the microtic group compared to the normal controls (p<0.05). Several complementary target messenger RNAs (mRNAs) had been predicted. OSR1, the target gene of miR-451 and miR-200c, was significantly up-regulated (p<0.01); TRPS1, the target gene of miR-200c, was significantly down-regulated in the microtic group compared to the controls (p<0.0001). CONCLUSION: The reduction in miR-200c expression and the accretion of miR-451 and miR-486-5p expression in microtic samples could be possible causes of the abnormal development of the external ear. OSR1 and TRPS1, as the complementary target mRNAs, may play important roles during the development of the external ear. Further studies are still needed to identify the miRNA target genes and to determine their function in the pathogenesis of microtia. This is the first report of a relationship between miRNAs and microtia.

Characteristic face: a key indicator for direct diagnosis of 22q11.2 deletions in Chinese velocardiofacial syndrome patients.

Velocardiofacial syndrome (VCFS) is a disease in human with an expansive phenotypic spectrum and diverse genetic mechanisms mainly associated with copy number variations (CNVs) on 22q11.2 or other chromosomes. However, the correlations between CNVs and phenotypes remain ambiguous. This study aims to analyze the types and sizes of CNVs in VCFS patients, to define whether correlations exist between CNVs and clinical manifestations in Chinese VCFS patients. In total, 55 clinically suspected Chinese VCFS patients and 100 normal controls were detected by multiplex ligation-dependent probe amplification (MLPA). The data from MLPA and all the detailed clinical features of the objects were documented and analyzed. A total of 44 patients (80.0%) were diagnosed with CNVs on 22q11.2. Among them, 43 (78.2%) presented with 22q11.2 heterozygous deletions, of whom 40 (93.0%) had typical 3-Mb deletion, and 3 (7.0%) exhibited proximal 1.5-Mb deletion; no patient was found with atypical deletion on 22q11.2. One patient (1.8%) presented with a 3-Mb duplication mapping to the typical 3-Mb region on 22q11.2, while none of the chromosomal abnormalities in the MLPA kit were found in the other 11 patients and 100 normal controls. All the 43 patients with 22q11.2 deletions displayed characteristic face and palatal anomalies; 37 of them (86.0%) had cognitive or behavioral disorders, and 23 (53.5%) suffered from immune deficiencies; 10 patients (23.3%) manifested congenital heart diseases. Interestingly, all patients with the characteristic face had 22q11.2 heterozygous deletions, but no difference in phenotypic spectrum was observed between 3-Mb and 1.5-Mb deletions. Our data suggest that the characteristic face can be used as a key indicator for direct diagnosis of 22q11.2 deletions in Chinese VCFS patients.

LINE-1 methylation status and its association with tetralogy of fallot in infants.

BACKGROUND: Methylation levels of long interspersed nucleotide elements (LINE-1) are representative of genome-wide methylation status and play an important role in maintaining genomic stability and gene expression. To derive insight into the association between genome-wide methylation status and tetralogy of fallot (TOF), we compared the methylation status of LINE-1 element between TOF patients and controls. The methylation of the NKX 2-5, HAND 1, and TBX 20 promoter regions was also evaluated. METHODS: Genomic DNA from right ventricular tissue samples was obtained from 32 patients with TOF and 15 control subjects. Sequenom MassARRAY platform was performed to examine the methylation levels of LINE-1, NKX2-5, HAND1 and TBX20. Mann-Whitney U test was used to compare differences in methylation levels between two groups. RESULTS: The methylation level of LINE-1 was significantly lower in patients with TOF, with a median of 57.95% (interquartile range [IQR]: 56.10%-60.04%), as opposed to 59.70% in controls (IQR: 59.00%-61.30%; P = 0.0021). The highest LINE-1 methylation level was 61.3%. The risk of TOF increased in subjects with the lowest methylation levels (less than or equal to 59.0%; OR = 14.7, 95% CI: 1.8-117.7, P = 0.014) and in those with medium methylation levels (59.0%-61.3%; OR = 2.0, 95% CI: 0.3-14.2, P = 0.65). An ROC curve analysis showed a relatively high accuracy of using the LINE-1 methylation level in predicting the presence of TOF (AUC = 0.78, 95% CI: 0.65-0.91; P = 0.002). The association of the LINE-1 methylation level with TOF was only observed in males (P = 0.006) and not in females (P = 0.25). Neither age nor gender was found to be associated with the LINE-1 methylation level in patients or controls. Higher methylation levels of NKX2-5 and HAND1 and lower methylation levels of TBX20 were also observed in patients with TOF than in controls. No association was found between the methylation levels of NKX2-5, HAND1 and TBX 20 with the LINE-1 methylation level. CONCLUSIONS: Lower LINE-1 methylation levels are associated with increased risk of TOF and may provide important clues for the development of TOF.