ATDC binds to KEAP1 to drive NRF2-mediated tumorigenesis and chemoresistance in pancreatic cancer.

Pancreatic ductal adenocarcinoma is a lethal disease characterized by late diagnosis, propensity for early metastasis and resistance to chemotherapy. Little is known about the mechanisms that drive innate therapeutic resistance in pancreatic cancer. The ataxia-telangiectasia group D-associated gene (ATDC) is overexpressed in pancreatic cancer and promotes tumor growth and metastasis. Our study reveals that increased ATDC levels protect cancer cells from reactive oxygen species (ROS) via stabilization of nuclear factor erythroid 2-related factor 2 (NRF2). Mechanistically, ATDC binds to Kelch-like ECH-associated protein 1 (KEAP1), the principal regulator of NRF2 degradation, and thereby prevents degradation of NRF2 resulting in activation of a NRF2-dependent transcriptional program, reduced intracellular ROS and enhanced chemoresistance. Our findings define a novel role of ATDC in regulating redox balance and chemotherapeutic resistance by modulating NRF2 activity.

Recombination of variable and host range regions of glycoprotein gp85 in different avian leukosis virus subgroup K isolates.

Given the high prevalence of avian leukosis virus subgroup K (ALV-K) in chickens in China, the positive rate of ALV-K in local chickens in Henan province was investigated, and the genetic region encoding the glycoprotein gp85 of isolates from positive chickens was analyzed. The positive rate of ALV-K in local chickens in Henan was found to be 87.2% (41/47). Phylogenetic analysis of gp85 sequences revealed six clusters that differed in their host range regions (hr1 and hr2) and variable regions (vr1, vr2, and vr3). Evidence of recombination of hr1, hr2, vr1, vr2, and vr3 was observed between the different clusters. The isolate HN23LS02 appears to have obtained its hr1 and hr2 regions from separate lineages via recombination but without having a significant affect on the replication capacity of the virus.

Improper toy waste handling can harm human health via seafood consumption: A comprehensive risk assessment of heavy metals.

Toy production has been increasing over the last few decades to meet the growing demands for toys across the globe, which has inevitably worsened the problem of toy waste. Given the lack of modern waste disposal facilities, rural villagers in developing countries often discard and incinerate toy waste in backyards or riverbanks, which may release the pollutants from toys (e.g., heavy metals), contaminate the surrounding areas, and eventually threaten the health of residents. As such, this study assessed the impact of improper toy waste handling on the nearby aquaculture site by measuring the contamination level of heavy metals (As, Cd, Cu, Pb, and Zn) in the sediment and seafood (crabs, shrimps, and fish). The health risk of heavy metals via seafood consumption was assessed in different groups of people (males, females, teenagers, and seniors). Results showed that the sediment and seafood at the aquaculture site were generally not contaminated with heavy metals (i.e., contamination factor and bioaccumulation factor < 1). However, consuming the seafood cultured at this site, especially for crabs, could pose health risks to humans due to As and Cd (hazard quotient > 1), irrespective of their age and gender. This risk could not be lowered by cooking, except As by boiling. Overall, we revealed that improper toy waste handling did not cause severe heavy metal pollution in the surrounding environment. However, the consumption of contaminated seafood could still threaten human health. To safeguard public health, we propose that toy waste be appropriately treated by the authorities concerned. At the same time, consuming seafood with long culture periods (e.g., crabs) should be reduced to minimize the dietary intake of heavy metals and their associated health risk.

Simultaneous determination of poly- and perfluoroalkyl substances and organophosphorus flame retardants in serum by ultra-performance liquid chromatography/tandem mass spectrometry.

RATIONALE: Poly- and perfluoroalkyl substances (PFASs) and organophosphorus flame retardants (OPFRs) are two types of emerging organic pollutants with potential human health hazards. Here, a rapid and sensitive method was developed for the determination of sixteen PFASs and seven OPFRs in human serum by ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS). METHODS: After optimizing the chromatographic and mass spectrometric conditions, respectively, 100 µL of serum sample was liquid-liquid extracted using 8 mL of methyl tert-butyl ether. The 23 targets were quantified within 8 min. All targets were quantified by the isotope-internal standard method in both negative- and positive-ion mode by UPLC/MS/MS. RESULTS: The method was validated in terms of sensitivity, linearity, precision, and accuracy. The limit of detection ranged between 0.004 and 0.650 ng/mL. Recoveries ranged from70.0% to 118.9% with a relative standard deviation lower than 20%. The developed method was successfully applied to analyze targeted analytes in human serum samples. A total of 13 of 23 analytes were detected in over 50% of samples. CONCLUSIONS: A sensitive and rapid method was developed to quantify sixteen PFASs and seven OPFRs in serum. Sensitivity, linearity, recovery, and precision were validated and found to be satisfactory. This method can be a valuable tool for evaluation of exposure to both PFASs and OPFRs with high separation efficiency and sensitivity.

Prenatal Exposure of Organophosphate Esters and Its Trimester-Specific and Gender-Specific Effects on Fetal Growth.

The toxicity of organophosphate esters (OPEs) on embryonic development is well noted in animal experiments, but epidemiological studies are still lacking. This study evaluated the prenatal exposure of OPEs and its trimester-specific and gender-specific effects on fetal growth. The correlations between OPE exposure and fetal growth were investigated by linear mixed-effect models and multivariable linear regression analyses. Prenatal exposure to tributyl phosphate (TBP) was negatively associated with a z-score of fetal abdominal circumference (AC), biparietal diameter (BPD), femur length (FL), and head circumference (HC). In the second trimester, the serum concentration of TBP was inversely related to the z-score of AC, BPD, and HC. In the third trimester, serum concentration of TBP was inversely related to AC, BPD, and FL z-scores. Prenatal exposure to tri-m-cresyl phosphate (TMCP) was inversely related to the z-score of AC, BPD, and HC. In the second trimester, TMCP was negatively correlated with AC, BPD, FL, and HC z-scores. After stratification by gender, male fetuses were more sensitive to OPE exposure. The above results remained robust after excluding pregnant women who gave preterm birth or those with low or high pre-pregnancy BMI. Our findings suggested that health effects of typical OPEs, particularly TBP and TMCP, should be taken into consideration in future works.

PM2.5 exposure associated with prenatal anxiety and depression in pregnant women.

BACKGROUND: Associations of air pollution with anxiety and depression were found in previous studies. However, whether air pollution exposure during pregnancy contributes to prenatal anxiety and depression or not is under-investigated. In this study, we aimed to analyze associations between fine particulate matter (PM2.5) exposure with anxiety and depression during pregnancy and to explore the critical window of PM2.5 exposure. METHODS: This study was based on the Shanghai Maternal-Child Pairs Cohort (Shanghai MCPC). We used a gap-filling random forest model to estimate PM2.5 exposure concentration during pregnancy of each participant. The Self-Rating Anxiety Scale (SAS) and the Center for Epidemiological Survey-Depression Scale (CES-D) were used to quantify the anxiety and depression levels in late pregnancy. Covariate information was obtained from medical records and questionnaires. We performed generalized linear regression and logistic regression models to assess the association and the critical window. RESULTS: Totally 3731 pregnant women were included, with the age of 28.85 ± 3.97 years old. Anxiety and depression rates were 10.8 % and 11.5 % respectively, according to the cut-off value of SAS and CES-D. Generalized linear regression results showed that the increase of PM2.5 concentration in three stages (gestational 0-13 weeks, 0-26 weeks, 0-36 weeks) was related to the increase of scale score. The PM2.5 concentration in 0-13 weeks could increase the risk of anxiety and depression by approximately 23 % and 25 %, respectively. And the gestational weeks 4th-13th were the suspicious critical window of PM2.5 exposure. CONCLUSION: The increased risk of anxiety or depression was related to PM2.5 exposure during pregnancy, especially early pregnancy.

[Oocyte maturation arrest due to compound heterozygous variants of the PATL2 gene in a case].

OBJECTIVE: To explore the genetic etiology of a patient with primary infertility and repeated failure of assisted reproductive technology. METHODS: Peripheral blood samples of the patient and her husband were collected for the extraction of genomic DNA and clinical exome sequencing. Candidate variants were verified by Sanger sequencing. RESULTS: The patient was found to harbor compound heterozygous variants of the PATL2 gene, namely c.223-14_223-2del and c.1369G>T (p.G457*). Sanger sequencing has verified that they were respectively inherited from her father and mother. The patient was diagnosed with oocyte maturation defect type 4. CONCLUSION: Oocyte maturation arrest due to mutations of the PATL2 gene can result in primary female infertility. Discovery of the novel c.1369G>T (p.G457*) variant has expanded the spectrum of pathogenic variants of the PATL2 gene.

A Metabolite Array Technology for Precision Medicine.

The application of metabolomics in translational research suffers from several technological bottlenecks, such as data reproducibility issues and the lack of standardization of sample profiling procedures. Here, we report an automated high-throughput metabolite array technology that can rapidly and quantitatively determine 324 metabolites including fatty acids, amino acids, organic acids, carbohydrates, and bile acids. Metabolite identification and quantification is achieved using the Targeted Metabolome Batch Quantification (TMBQ) software, the first cross-vendor data processing pipeline. A test of this metabolite array was performed by analyzing serum samples from patients with chronic liver disease (N = 1234). With high detection efficiency and sensitivity in serum, urine, feces, cell lysates, and liver tissue samples and suitable for different mass spectrometry systems, this metabolite array technology holds great potential for biomarker discovery and high throughput clinical testing. Additionally, data generated from such standardized procedures can be used to generate a clinical metabolomics database suitable for precision medicine in next-generation healthcare.

An Optimized Weighted Naïve Bayes Method for Flood Risk Assessment.

Floods occur frequently and cause considerable damage to local environments. Effectively assessing the flood risk contributes to reducing loss caused by such disasters. In this study, the weighted naïve Bayes (WNB) method was selected to evaluate flood risk, and the entropy weight method was employed to compute the weights. A sampling and verifying model was employed to generate the most accurate conditional probability table (MACPT) to calculate the probability of flooding. When using the framework integrating WNB with the sampling and verifying model, previous studies could not obtain a WNB-based MACPT and the WNB classification accuracy, for lacking WNB functions that could be called directly. Facing this issue, in this study we developed WNB functions with the MATLAB platform to directly integrate with the sampling and verifying model to generate a WNB-based MACPT, contributing to the greater interpretability and extensibility of the model. Shantou and Jieyang cities in China were selected as the study area. The results demonstrate that: (1) a WNB-based MACPT can reflect the real spatial distribution of flood risk and (2) the WNB outperform the NB when integrated with the sampling and verifying model. The resulting gridded estimation reveal a detailed spatial pattern of flood risk, which can serve as a realistic reference for decision making related to floods. Furthermore, the proposed method uses less data, which would be helpful in developing countries where long-term intensive hydrologic monitoring is limited.

Paraben Exposure Related To Purine Metabolism and Other Pathways Revealed by Mass Spectrometry-Based Metabolomics.

Parabens are widely used as common preservatives in the pharmaceutical and cosmetic industries. Exposure to parabens has been found to be associated with metabolic alterations of human and an increased risk of metabolic disease, such as diabetes. However, limited information is available about metabolic pathways related to paraben exposure. In this study, three parabens were determined in the urine samples of 88 pregnant women by using ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QqQ MS). The samples were divided into different groups based on tertile distribution of urinary paraben concentrations. Metabolic profiling of the 88 urine samples was performed by using UHPLC coupled with Orbitrap high-resolution MS. Differential metabolites were screened by comparing the profiles of urine samples from different paraben-exposure groups. The identified metabolites included purines, acylcarnitines, etc., revealing that metabolic pathways such as purine metabolism, fatty acid ß-oxidation, and other pathways were disturbed by parabens. Eighteen and three metabolites were correlated (Spearman correlation analysis, p < 0.05) with the exposure levels of methyparaben and propylparaben, respectively. This is the first MS-based nontargeted metabolomics study on pregnant women with paraben exposure. The findings reveal the potential health risk of exposure to parabens and might help one to understand the link between paraben exposure and some metabolic diseases.

Association between phthalate exposure and blood pressure during pregnancy.

BACKGROUND: Phthalates are endocrine disrupting chemicals (EDCs) that pose a serious hazard to the human health. Many epidemiological studies revealed a relationship between phthalates exposure and blood pressure in general population, while the relationship in pregnant women remains unknown. OBJECTIVES: Aimed to elucidate whether phthalate exposure is associated with blood pressure among pregnant women. METHODS: This study included 636 participants from Wuhan, China. Urine samples were conducted repeatedly in three trimesters, and 9 phthalates were measured in these samples. After each urine was sampled, all the participants completed blood pressure measurements. Associations between repeated measurements of phthalate concentration and blood pressure were evaluated by using generalized estimating equations. Stratified analysis by fetus gender was conducted. RESULTS: Among the pregnant women with male fetuses, mono-i-butyl phthalate (MiBP) exposed in the 1st trimester was associated with the increased diastolic blood pressure (DBP) measured in the 2nd trimester, while the environmental risk score (ERS) measured in the 1st and 2nd trimester was positively associated with systolic blood pressure (SBP) and DBP in the 2nd trimester. No significant relationships were observed among all the population or pregnant women with female fetuses. CONCLUSIONS: Exposure to higher levels of MiBP may be related to increased blood pressure during pregnancy in pregnant women with male fetuses.

A novel optimized repeatedly random undersampling for selecting negative samples: A case study in an SVM-based forest fire susceptibility assessment.

The negative sample selection method is a key issue in studies of using machine learning approaches to spatially assess natural hazards. Recently, a Repeatedly Random Undersampling (RRU) was proposed to address the randomness problem faced in Single Random Sampling. However, the RRU cannot guarantee that the generated classifier has the best classification performance during the repeatedly random sampling process. To address this weakness, in this study we proposed an optimized RRU, which follows the idea of RRU, and then changing its rule to find a best classifier. Then, the selected classifier, the actual most accurate classifier (MAC), was employed to compute the probability of hazard occurrence. Support Vector Machine (SVM) was selected as the analysis method, and Genetic Algorithm was employed to compute the parameters of SVM. Forest fire susceptibility was assessed in Huichang County in China due to its forest values and frequent fire events. The results indicated that compared with the RRU, the optimized RRU can find out an actual MAC which has the best classification performance among possible MACs; also, the fire susceptibility map generated by the actual MAC comforts to objective facts. The generated fire susceptibility map can provide useful decision supports for local government to reduce forest fire risks. Moreover, the proposed sampling method, the optimized RRU, presented an enhanced approach for selecting negative samples, which makes the results of forest fire susceptibility assessment more reliable and accurate.

Exposure Assessment of Bisphenols in Chinese Women during Pregnancy: A Longitudinal Study.

Bisphenol S (BPS) and bisphenol F (BPF) are increasingly used in manufacturing consumer products to replace the use of bisphenol A (BPA), but exposure data are limited, particularly among pregnant women. Here, we measured BPA, BPS, and BPF levels in urine samples, collected from 941 pregnant women over three trimesters. We examined the correlations, coexposure patterns, variability, and predictors of bisphenols using Spearman's correlation coefficient, percentile analysis, intraclass correlation coefficient, and linear mixed models, respectively. We assessed health risks using average concentrations of bisphenols over three trimesters. The three bisphenols were detected in more than 50% of samples, among which BPA was the predominant one. Cashiers, office workers, teachers, and salespersons had elevated urinary BPS concentrations, while healthcare workers had relatively higher BPA concentrations. About 15 participants had potential health risks induced by exposure to bisphenol mixtures. These findings indicate that exposure to multiple bisphenols at low levels is common over three trimesters. Multiple measurements of urinary BPA and BPS concentrations are needed for more accurate evaluation of the exposure levels during pregnancy, while urinary BPF concentrations during pregnancy are moderately reliable. Occupational exposure should be taken into consideration in future demographic studies.

Variations, Determinants, and Coexposure Patterns of Personal Care Product Chemicals among Chinese Pregnant Women: A Longitudinal Study.

Exposure to mixtures of personal care product chemicals (PCPCs) is commonplace among the Chinese population; yet, limited data are available on the variations, determinants, and coexposure patterns of PCPCs, particularly among pregnant women at multiple time points during gestation. Here, we measured concentrations of 11 most common PCPCs (five parabens, five benzophenones, and triclosan) in 2823 urine samples collected from 941 pregnant women over three trimesters. Based on the quantification results, we calculated the intraclass correlation coefficient (ICC) to assess within-person variability of targeted compounds, applied linear mixed mode models to explore associations between urinary concentrations of PCPCs and exposure-related factors, and used percentile analysis to evaluate exposure to specific or multiple chemicals at one or three trimesters. Seven targeted compounds: methylparaben (MeP), ethylparaben (EtP), propylparaben (PrP), 4-hydroxybenzophenone (4-OH-BP), 2,4-dihydroxybenzophenone (BP-1), 2-hydroxy-4-methoxybenzophenone (BP-3), and triclosan (TCS) were detected in over 66% of samples. The median urinary concentrations (ng/mL) of MeP, EtP, PrP, 4-OH-BP, BP-1, BP-3, and TCS were 15.44, 0.49, 0.61, 0.16, 0.25, 0.53, and 0.48, respectively. We observed that benzophenones (ICC: 0.46-0.55) and triclosan (ICC: 0.50) were less variable than parabens (ICC: 0.35-0.40). Urinary levels of parabens were related to physical activity frequency; urinary levels of benzophenones were associated with the refurbishment of homes and household income, and urinary levels of triclosan were contingent upon the personal basic information (prepregnancy body mass index and age). Notably, higher levels of benzophenones and triclosan but lower paraben levels were observed in summer than in winter. Both coexposure to high percentiles of multiple pollutants at one trimester and exposure to one pollutant at high-dose through three trimesters were rare in the study population. Our findings suggest that these exposure-related factors should be taken into consideration, and health risks should be assessed on mixtures of pollutants in future epidemiological studies.

Repeated Measurements of Paraben Exposure during Pregnancy in Relation to Fetal and Early Childhood Growth.

Parabens are potential endocrine disruptors with short half-lives in the human body. To date, few epidemiological studies regarding repeated paraben measurements during pregnancy associated with fetal and childhood growth have been conducted. Within a Chinese prenatal cohort, 850 mother-infant pairs from whom a complete set of maternal urine samples were acquired during three trimesters were included, and the levels of five parabens were measured. We assessed the associations of both average and trimester-specific urinary paraben levels with weight and height z-scores at birth, 6 months, 1, and 2 years of age. In all infants, each doubling increase in average ethyl paraben (EtP) was associated with -2.82% (95% CI: -5.11%, -0.53%) decrease in weight z-score at birth, whereas no significant age-specific associations were identified. After stratifying by sex, we further observed age-specific association of average EtP with -3.96% (95% CI: -7.03%, -0.89%) and -3.38% (95% CI: 6.72%, -0.03%) reduction in weight z-scores at 1 and 2 years in males, respectively. Third-trimester EtP was negatively associated with weight z-scores at birth, 1 and 2 years in males. Our results suggested negative associations between prenatal paraben exposure and fetal and childhood growth, and the third trimester may be the window of susceptibility.

Determinants of exposure levels, metabolism, and health risks of phthalates among pregnant women in Wuhan, China.

Concerns on minimizing health risks of phthalates have been raised due to their widespread exposure and well-documented endocrine disrupting properties, but the determinants of levels, metabolism, and health risks of phthalate exposures have not been thoroughly characterized among the Chinese population, particularly pregnant women. The metabolites of five phthalates were analyzed: diethyl (DEP), diisobutyl (DiBP), di-n-butyl (DnBP), di-(2-ethylhexyl) (DEHP), and benzyl butyl phthalate (BBzP) were analyzed in urine samples collected from 946 mothers in Wuhan during 2014-2015. We applied linear mixed models to investigate the relationships between biomarkers (e.g., urinary concentrations of phthalate metabolites, phthalates, and ratios of metabolites) and factors including sampling seasons and epidemiological characteristics. We calculated estimated daily intake (EDI) using average phthalate concentrations over three trimesters and hazard index (HI) by dividing EDI by tolerance daily intake. About 24.9% of participants were at health risks with HI > 1. The largest health risks were driven by one specific phthalate (DnBP or DEHP). We observed lower urinary levels of phthalate metabolites in winter. Elevated levels were found in mothers with higher education levels or those employed. Mothers who got pregnant on purpose had lower phthalate concentrations than those got pregnant by accident. More recent exposure to phthalates was observed among groups of mothers giving birth to girls, or those who got excessive gestational weight gain. Younger mothers were more susceptible to phthalate exposure. This repeated measurement study suggests that the intervention should be taken to limit application and production of DnBP and DEHP, and highlights that typical demographic factors should be taken into account in demographic studies.

Investigation on Metabolism of Di(2-Ethylhexyl) Phthalate in Different Trimesters of Pregnant Women.

Di(2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer but shows diverse toxicity. To investigate the time- and maternal age-differences in metabolism process of DEHP in pregnant women, three urine samples were collected from each pregnant woman ( n = 847) at the first (T1, mean 13.04 gestational weeks), the second (T2, mean 23.63 gestational weeks) and the third time point (T3, mean 35.91 gestational weeks), respectively. Four metabolites of DEHP were analyzed in 2541 urine samples (847 × 3) by using ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry. The level of urinary mono(2-ethylhexyl) phthalate (MEHP) had a decreasing trend across the pregnancy periods. The geometric mean concentrations of mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono(2-ethyl-5-oxohexyl) phthalate (MEOHP) and mono(2-ethyl-5-carboxypentyl) phthalate (MECPP) were significantly decreased in T2 than T1, and recovered slightly in T3. The transformation rate of MEHP to MEHHP in T3 was significantly higher than those in other two time points. The transformation rate of MEHHP to MEOHP in T2 and T3 was significantly higher than that in T1, indicating the oxidation was more efficient in late pregnancy compared with early and middle pregnancy. The percentages of oxidation products MEHHP and MECPP were higher in the higher-age group compared with the lower-age group in the second trimester samples. It is therefore concluded that DEHP metabolism may be influenced by the pregnancy stage and maternal age. The findings may benefit the risk assessment and toxicity evaluation of DEHP.

Determination of macrolide antibiotics residues in pork using molecularly imprinted dispersive solid-phase extraction coupled with LC-MS/MS.

A class-specific macrolide molecularly imprinted polymer was synthesized by precipitation polymerization using tulathromycin as the template and methacrylic acid as the functional monomer. The polymers revealed different specific adsorption and imprinting factor for macrolides with different spatial arrangement of side chains as well as lactonic ring size. And the molecularly imprinted polymer possessed maximum adsorption capacity (54.1 mg/g) and highest imprinting factor (2.4) toward 15-membered ring azithromycin. On the basis of molecularly imprinted polymer dispersive solid-phase extraction, a rapid, selective, and reproducible method for simultaneous determination of seven macrolide antibiotics residues in pork was established by using liquid chromatography with tandem mass spectrometry. At spiking levels of 5, 10, 25, and 100 µg/kg, average recoveries of seven macrolides ranged from 68.6 to 95.5% with intraday and interday relative standard deviations below 8%. The limits of detection and limits of quantification were 0.2-0.5 and 0.5-2.0 µg/kg, respectively.

Determination of Ten Macrolide Drugs in Environmental Water Using Molecularly Imprinted Solid-Phase Extraction Coupled with Liquid Chromatography-Tandem Mass Spectrometry.

With the extensive application of antibiotics in livestock, their contamination of the aquatic environment has received more attention. Molecularly imprinted polymer (MIP), as an eco-friendly and durable solid-phase extraction material, has shown great potential for the separation and enrichment of antibiotics in water. This study aims at developing a practical and economical method based on molecularly imprinted solid phase extraction (MISPE) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for simultaneously detecting ten macrolide drugs in different sources of water samples. The MIP was synthesized by bulk polymerization using tylosin as the template and methacrylic acid as the functional monomer. The MIP exhibited a favorable load-bearing capacity for water (>90 mL), which is more than triple that of non-molecularly imprinted polymers (NIP). The mean recoveries of macrolides at four spiked concentration levels (limit of quantification, 40, 100, and 400 ng/L) were 62.6⁻100.9%, with intra-day and inter-day relative standard deviations below 12.6%. The limit of detection and limit of quantification were 1.0⁻15.0 ng/L and 3.0⁻40.0 ng/L, respectively. Finally, the proposed method was successfully applied to the analysis of real water samples.

[Non-invasive prenatal diagnosis for beta-thalassemia by detecting paternal CD41-42 mutation in cell-free DNA derived from maternal plasma with droplet digital PCR].

OBJECTIVE: To establish a non-invasive method for beta-thalassemia by detecting parental CD41-42 mutation in cell-free DNA derived from maternal plasma with droplet digital PCR (ddPCR). METHODS: Beta-actin gene and beta-thalassemia gene CD41-42 mutation were respectively set as the reference and target sequences. A novel method was established based on Bio-Rad ddPCR technique with specific primers and TaqMan probes for the two genes. The accuracy, sensitivity and detective linearity range of the developed method were evaluated by detection of the target gene gradient concentration samples. The applicability was also evaluated by testing 20 maternal plasma samples. RESULTS: The ddPCR method could accurately detect the beta-thalassemia CD41-42 mutation in cell-free DNA derived from maternal plasma. Within the target sequence concentration ratio of 5.00%-0.50%, the relative errors were all < 0.05, the linear regression equation was Y=1.0101-X-0.0071 and R2=0.9994. The results of 20 maternal plasma cell-free DNA samples were all consistent with those of the follow-up testing. CONCLUSION: A ddPCR method for detecting parental CD41-42 mutation in cell-free DNA from maternal plasma was developed. The method is simple, rapid, accurate, and can be applied for non-invasive prenatal diagnosis for couples simultaneously carrying the CD41-42 mutation.