Noninvasive prenatal diagnosis based on cell-free DNA for tuberous sclerosis: A pilot study.

BACKGROUND: Noninvasive prenatal diagnosis (NIPD) based on cell-free DNA (cfDNA) has been introduced into the clinical application for some monogenic disorders but not for tuberous sclerosis (TSC) yet, which is an autosomal dominant disease caused by various variations in TSC1 or TSC2 gene. We aimed to explore the feasibility of NIPD on TSC. METHODS: We recruited singleton pregnancies at risk of TSC from 14 families with a proband child. Definitive NIPD for TSC was performed using targeted next-generation sequencing of cfDNA in parallel with maternal white blood cell DNA (wbcDNA). The NIPD results were validated by amniocentesis or postnatal gene testing and follow-up of the born children. RESULTS: Missense mutations, nonsense mutations, frameshift mutations, and splice-site variants which were obtained through de-novo, maternal, or paternal inheritance were included. The mean and minimum gestational weeks of NIPD were 17.18 ± 5.83 and 8 weeks, respectively. The NIPD results were 100% consistent with the amniocentesis or postnatal gene testing and follow-up of the born children. CONCLUSION: This study demonstrates that NIPD based on cfDNA is feasible for TSC, but required to be confirmed with more samples. Studies on TSC can contribute to the application and promotion of NIPD for monogenic disorders.

[Preparation and in vitro evaluation of arsenic trioxide glioma targeting drug delivery system loaded by PAMAM dendrimers co-modified with RGDyC and PEG].

Arsenic trioxide (ATO) is an effective component of traditional Chinese medicine arsenic. The existing studies have shown its good inhibition and apoptosis ability on a variety of tumours. However, its toxicity and difficulties in the permeability into the blood brain barrier (BBB) has the limitation in the application of glioma treatment. Polyamide-amine dendrimer (PAMAM) is a synthetic polymer with many advantages, such as a good permeability, stability and biocompatibility. Additionally, the 5th generation of PAMAM is an ideal drug carrier due to its three-dimensional structure. In this study, the 5th generation of PAMAM co-modified with RGDyC and PEG, then confirmed by ¹H-NMR. The average particle size of nanoparticles was about 20 nm according to the nanoparticle size-potential analyser and transmission electron microscopy. in vitro release showed that the nanocarrier not only has the sustained release effect, but also some pH-sensitive properties. The cell results showed that PAMAM co-modified with RGDyC and PEGAM has a lower cytotoxicity than the non-modified group in vitro. Accordingly, the drug delivery system has a better anti-tumour effect across the blood brain barrier (BBB) in vitro, which further proves the tumour targeting of RGDyC.

Expressions of programmed death (PD)-1 and PD-1 ligand (PD-L1) in cervical intraepithelial neoplasia and cervical squamous cell carcinomas are of prognostic value and associated with human papillomavirus status.

AIM: The programmed death 1/programmed death 1 ligand (PD-1/PD-L1) pathway can decrease the immune clearance effects of antigen-presenting cells and T lymphocytes to promote immune evasion of cervical cancer cells. However, the effects of this pathway on cervical intraepithelial neoplasia (CIN) progression and squamous cell carcinoma (SCC) metastasis are not clear. We herein investigated whether human papillomavirus infection could affect PD-1 and PD-L1 expression in CIN, and whether their expression is associated with CIN progression and SCC metastasis. METHODS: We collected paraffin-embedded samples from two cohorts of patients: (i) CIN samples from cohort I (40 women who tested positive or negative for high-risk human papillomavirus [HR-HPV] with grades 0, I, and II-III CIN); and (ii) paired primary and metastatic tumor samples from cohort II (20 SCC patients with or without metastasis). Immunohistochemistry was used to detect expressions of PD-L1 in tumor cells and PD-1 in tumor-associated macrophages and tumor-infiltrating lymphocytes. We also measured P16INK4a expression and interferon-γ levels in the cervical tissues. RESULTS: The most common HPV type seen in both cohorts of patients was HPV16, followed by HPV18. Increase in PD-L1 and PD-1 expression was positively correlated with HPV-positivity, increase in CIN grade, and tumor metastasis. Furthermore, upregulation of the PD-1/PD-L1 pathway was associated with decreased expression of the pro-inflammatory cytokine, interferon-γ and increased expression of P16INK4a . CONCLUSION: Expression of PD-L1 and PD-1 could be used as clinical prognostic biomarkers for evaluating CIN and cervical cancer because of its positive correlation with CIN progression and tumor metastasis.

The Association of Maternal Body Composition and Dietary Intake with the Risk of Gestational Diabetes Mellitus during the Second Trimester in a Cohort of Chinese Pregnant Women.

OBJECTIVE: To investigate the association of maternal body composition and dietary intake with the risk of gestational diabetes mellitus (GDM). METHODS: A total 154 GDM subjects and 981 controls were enrolled in a prospective cohort study in 11 hospitals from May 20, 2012 to December 31, 2013. Bioelectrical impedance analysis and dietary surveys were used to determine body composition and to evaluate the intake of nutrients in subjects at 21-24 weeks' gestation (WG). Logistic regression analysis was applied to explore the relationships of maternal body composition and dietary intake with the risk of GDM morbidity. RESULTS: Age, pre-pregnant body weight (BW), and body mass index (BMI) were associated with increased risk of GDM. Fat mass (FM), fat mass percentage (FMP), extracellular water (ECW), BMI, BW, energy, protein, fat, and carbohydrates at 21-24 WG were associated with an increased risk of GDM. In contrast, fat free mass (FFM), muscular mass (MM), and intracellular water (ICW) were associated with a decreased risk of GDM. CONCLUSION: Maternal body composition and dietary intake during the second trimester of pregnancy were associated with the risk of GDM morbidity.

[Correlation between primary cilium and Wnt signaling pathway].

Primary cilium is a microtubule-based organelle,which develops from the mother centriole of the centrosome. It is an antenna-like structure that anchors at the cell membrance, protruding from the cell surface. Primary cilium acts as a sensory organelle that receives different kinds of signals from the environment and transmits signals to cells to elicit cellular responses. Recent studies have revealed that primary cilium play an important role in transmitting Wnt signaling, which is critical for embryonic development. Dysfunction of primary cilium deregulates Wnt signaling, causing a series of pathological changes in different organs of the embryo, resulting in ciliopathies. In this review, we summarize correlation among primary cilium,Wnt/ß-catenin signaling,Wnt/PCP signaling and ciliopathies. Current therapies in ciliopathies are also discussed. Highlights on these researches will encourage the development of Wnt-associated diagnostic tools and therapy for ciliopathies.

[Prenatal genetic test and clinical guidance for 213 hereditary deaf families].

OBJECTIVE: To summarize the workflow, strategy and experience of prenatal genetic test for deafness based on the 6-year clinical practice. METHODS: There were 213 families who received prenatal test from 2005 to 2011. Among the 213 families, 205 families had had one deaf child, including 204 couples with normal hearing and one couple of the deaf husband and normal wife, 8 families including 6 couples with normal hearing and 2 deaf couples, had no child before test. Genomic and mitochondrial DNA of each subject was extracted from whole blood. The etiology and recurrent risks in 212 families were confirmed by means of the genetic test of GJB2, SLC26A4 and mtDNA 12sRNA, but one family carried POU3F4 c.647G > A heterozygous mutation causing X-linked hereditary hearing impairment confirmed by pedigree study. The prenatal test was carried out during the pregnancy of all mothers from 11 to 30 weeks, and the following genetic information and counseling were supplied based on the results. RESULTS: The recurrent risk was 25% in 209 families, including 204 families with one deaf child and 5 families without child, among which all couples were GJB2 or SLC26A4 mutation carriers and deaf children were caused by homozygous or compound GJB2/SLC26A4 mutations; The recurrent risk was 50% in 3 families, the father and his child in one family had compound SLC26A4 mutations and the mother with heterozygous SLC26A4 mutation, the wife had POU3F4 c.647G > A heterozygous mutation in another one family, and the husband with compound SLC26A4 mutations and the wife with mtDNA A1555G mutation and heterozygous SLC26A4 mutation simultaneously happened in the rest one family; The recurrent risk was 100% in one family of the deaf couple who were both found to carry homozygous or compound GJB2 mutations, and the deaf wife got pregnant by artificial insemination with the sperm from the local Human Sperm Bank. 226 times of prenatal test were applied in all 213 families that 11 families of them received prenatal test twice, and one family received three times. 46 times of prenatal testing showed that the fetuses carried parental mutations simultaneously or the same mutations with probands; while 180 times of prenatal test showed that the fetuses carried only one parental mutation or did not carry any mutation from parents. The following visit showed that all of these 180 families had given birth to babies who were all revealed to have normal hearing by new born hearing screening test. CONCLUSIONS: Prenatal diagnosis for deafness assisted by genetic test can provide efficient information about offspring's hearing condition, and the normative workflow and precise strategy highly guarantee the safe and favorable implementation of prenatal diagnosis.

[Value of detection of cell-free fetal DNA in maternal plasma in the prenatal diagnosis of chromosomal abnormalities].

OBJECTIVE: To investigate the value of detection of fetal cell-free fetal DNA (cff-DNA) in maternal plasma in the prenatal diagnosis of chromosomal abnormalities. METHODS: The plasma from 3200 gravidas (singleton with 20.3 ± 3.8 gestational weeks) was collected from April 1(st) 2011 to May 30(th) 2012. They were divided into 3 groups: (1) To tally 1720 cases were included in the high-risk serological screening group, in which women were younger than 35 years and got high-risk results in serological screening; (2) To tally 1310 cases were included in the advanced age group, in which women's age was more than 35 years; (3) To tally 170 cases were included in the supplementary group, in which women were younger than 35 years and got low-risk results in serological screening, or women who didn't take serological screening tests. All the 3030 gravidas in group 1 and 2 didn't take invasive prenatal diagnosis because of fear of abortion or short of prenatal diagnosis. Cff-DNA were detected by next generation sequencing in Shenzhen BGI Genomics Center for clinical laboratory. Amniocentesis and karyotype analysis were provided to the positive cases and women with negative results were followed-up by telephone. RESULTS: (1) The 3200 cases took cff-DNA detection, and 31 cases got positive results, including 27 cases of trisomy 21 and 4 cases of trisomy 18. Sixteen cases of trisomy 21 and 1 case of trisomy 18 were in the high-risk serological screening group. 7 cases of trisomy 21 and 2 cases of trisomy 18 were in the advanced age group. Four cases of trisomy 21 and 1 case of trisomy 18 were in the supplementary group. (2) And the 84% (26/31) cff-DNA detecting positive cases received amniocentesis. In the 27 trisomy 21 positive cases, 23 received amniocentesis and got karyotype of 47XN, +21, with the diagnostic accordance rate of 100%. In the 4 cases who didn't take karyotype analysis, fetal anomaly (ventricular septal defect, dextrocardia and choroid plexus cyst) was found in 1 case before 20 gestational weeks; intrauterine fetal demise happened in 1 case before getting the result; 2 other cases who already had healthy children took abortion in the local hospital without taking amniocentesis. In the 4 trisomy 18 positive cases, 3 took amniocentesis, 2 of which were trisomy 18 and took abortion, the other was chimera (46, XN/47, XN, +18) with only 2% cells of trisomy 18, with no malformation found after delivery. Hypoevolutism (3 weeks less than gestational week), general hydropsy and intrauterine fetal demise happened before the other case took amniocentesis. (3) Follow up of cff-DNA negative cases:until May 30(th) 2012, no Down's baby was found in the 1230 cases with cff-DNA test negative results. CONCLUSIONS: (1) The non-invasive fetal trisomy test (NIFTY) by next generation sequencing is a safe, accurate and high throughput method for the prenatal diagnosis of trisomy-21. (2) Use NIFTY as a further screening for pregnant women with high-risk serological screening results could lower invasive prenatal diagnosis rate. (3) Cases with positive NIFTY test results should receive amniocentesis and karyotype analysis to confirm the diagnosis before abortion.

Human amniotic fluid-derived stem cells can differentiate into hepatocyte-like cells in vitro and in vivo.

Although human amniotic fluid is an attractive source of multipotent stem cells, the potential of amniotic fluid stem cells (AFSCs) to differentiate into hepatic cells has not been extensively evaluated. In this study, we examined whether human AFSCs can differentiate into a hepatic cell lineage in vitro and in vivo. After being treated with cytokines (fibroblast growth factor 4, basic fibroblast growth factor, hepatocyte growth factor, and oncostatin), AFSCs developed a morphology similar to that of hepatocytes. RT-PCR and immunofluorescence analysis showed that the treated AFSCs expressed the hepatocyte-specific markers albumin, cytokeratin 18, and alpha-fetoprotein. The differentiated cells also developed hepatocyte-specific functions, i.e., they secreted albumin, absorbed indocyanine green, and stored glycogen. When transplanted into CCl(4)-injured immunodeficient mice, undifferentiated AFSCs were integrated into the liver tissue, and they expressed markers characteristic of mature human hepatocytes. Although integration of AFSCs into the liver was limited (0.1-0.3% of hepatocytes), histological analysis showed that the recipient mice recovered more rapidly from CCl(4) injury than CCl(4)-injured mice that did not receive AFSCs. AFSCs can differentiate into hepatocyte-like cells in vitro and in vivo and can represent an easily accessible source of progenitor cells for hepatocyte regeneration and liver cell transplantation.

[Detection of trisomy 21 by quantitative fluorescent PCR in clinical samples undergoing prenatal diagnosis for hereditary hearing loss].

OBJECTIVE: To establish the genetic test technique of trisomy 21 concurrently conducts with prenatal diagnosis for hereditary hearing loss. METHODS: Fifty-four pregnant women who underwent prenatal diagnosis for hearing loss of their fetuses in Chinese People's Liberation Army General Hospital from March 2009 to May 2010 were enrolled in this study. All probands from the deaf families have confirmed the causative mutation for hearing loss in Genetic Testing Center in Chinese People's Liberation Army General Hospital. The mean age of 54 pregnant women is 31 years at pregnancy of 18 - 26 weeks, 5 cases > pregnancy of 23 weeks, 9 cases ≥ 35 years. All subjects did not conduct the serologic tests for trisomy 21 before. Fifteen to twenty ml amniotic fluid was drawn from 49 cases at pregnancy of 18 - 23 weeks and 5 cases > pregnancy of 23 weeks. One to two ml umbilical blood was drawn from 5 cases > pregnancy of 23 weeks. For 9 cases ≥ 35 years, amniotic fluid cell culture and karyotyping analysis were conducted concurrently. A multiple quantitative fluorescent (QF) PCR and six microsatellite markers were applied to diagnosis trisomy 21. The samples with peaks of 1:1:1 or 2:1 at two microsatellite markers can be diagnosed as trisomy 21. RESULTS: (1) Fifty-four fetuses were successfully conducted prenatal genetic diagnosis for hearing loss (included GJB2 and SLC26A4). Ten fetuses copied the exactly same genotypes as the probands. The other 44 cases fetuses did not copy the same genotypes as the probands and won't develop hearing loss. The hearing test showed normal hearing for the neonates. (2) All the 54 fetuses were excluded of trisomy 21 by QF-PCR and were verified after birth. Five fetuses with advanced maternal age were performed karyotyping analysis and showed normal. The diagnostic results of QF-PCR can be obtained in 1-3 days without misdiagnosed. CONCLUSIONS: QF-PCR is an efficient, rapid and accurate technique for detection of trisomy 21 without increasing sample amount. It can be used for fetuses who were undertaken hearing loss gene test or other prenatal gene test.

[Development of multiple quantitative fluorescent PCR for rapid diagnosis of common aneuploidy and it's clinical application].

In this study we have established a technique of multiple quantitative fluorescent polymerase chain reaction (QF-PCR) for prenatal diagnosis of common chromosomal abnormality using multiple short tandem repeat markers (STR-marker) on chromosomes 21 and 18 with the DNA samples from 20 cases of Down's syndrome, 3 cases of trisomy 18 and 40 cases normal controls. The technique established was applied in prenatal diagnosis in 165 clinical cases and 4 cases of newborn infants with digestive tract obstruction. The result this technique was compared with the results of karyotyping. Four cases of trisomy 21 and 1 case of trisomy 18 were identified among 169 samples, which was completely concordant with the results of karyotyping. All clinical samples were diagnosed in 1-3 days without misdiagnosis and missed diagnosis. Five cases were diagnosed by QF-PCR only due to the failure of karyotyping. Twenty-two cases of fetuses with structure malformation indicated by B-ultrasonography were subjected to karyotyping. One case of 45, X and 1 case of 47, XXY were identified. In conclusion, QF-PCR technique is rapid and accurate for the detection of trisomy 21 and trisomy 18. It is suitable for prenatal diagnosis of common chromosomal abnormality for pregnant women with advanced ages who were identified as having a high risk by serum screening. QF-PCR technique combined with karyotyping can provide better service for clinical demanding of prenatal diagnosis.

Guar gum/ethylcellulose coated pellets for colon-specific drug delivery.

The aim of this work was to investigate guar gum/ethylcellulose mix coated pellets for potential colon-specific drug delivery. The coated pellets, containing 5-fluorouracil as a model drug, were prepared in a fluidized bed coater by spraying the aqueous/ethanol dispersion mixture of guar gum and ethylcellulose. The lag time of drug release and release rate were adjustable by changing the ratio of guar gum to ethylcellulose and coat weight gain. In order to find the optimal coating formulation that was able to achieve drug targeting to the colon, the effect of two independent variables (the ratio of guar gum to ethylcellulose and the coat weight gain) on drug release characteristics was studied using 3 x 4 factorial design and response surface methodology. Results indicated that drug release rate decreased as the proportion of ethylcellulose in the hybrid coat and the coat weight gain increased. When the ratio of guar gum to ethylcellulose was kept in the range of 0.2 to 0.7, and the coat weight gain in the range of 250% to 500%, the coated pellets can keep intact for about 5 h in upper gastrointestine and achieve colon-specific drug delivery. The pellets prepared under optimal conditions resulted in delayed-release sigmoidal patterns with T(5%) (time for 5% drug release) of 5.1 - 7.8 h and T(90%) (time for 90% drug release) of 9.8 - 16.3 h. Further more, drug release was accelerated and T(90%) of the optimum formulation pellets decreased to 9.0 - 14.5 h in pH 6.5 phosphate buffer with hydrolase. It is concluded that mixed coating of guar gum and ethylcellulose is able to provide protection of the drug load in the upper gastrointestinal tract, while allowing enzymatic breakdown of the hybrid coat to release the drug load in the colon.

[Preliminary study on xenotransfusion from porcine red blood cell into Rhesus monkey].

In order to study the possibility of xenotransfusion from porcine red blood cell (pRBC) to primate, the antigens on pRBC surface were modified to make it more compatible to primate sera. Porcine RBCs were subjected to both enzymatic removal of membrane alpha-Gal antigens with recombinant alpha-galactosidase (AGL) and covalent attachment of succinimid propionate-linked methoxypolyethyleneglycol (mPEG-SPA) to camouflage non-alphaGal antigens. The effects of double modifications were determinated by hemagglutination and clinical cross-match testing with rhesus sera. In vivo clearance rates and safety of modified pRBCs were measured after it was transfused into Rhesus monkey with or without immunosuppressant treatment. The validity of pRBC was detected in exsanguine Rhesus monkey model. The results showed that AGL could effectively remove alpha-Gal xenoantigens on pRBC membrane and reduce hemagglutination. The combination of mPEG modification with AGL treatment could significantly increased compatibility between pRBCs and Rhesus monkey sera. Modified pRBCs were detectable in Rhesus monkey blood at 12 hours after transfusion, and their survival time was 40 hours in the immunosuppressant-treated Rhesus monkey. In vivo survival rates of pRBCs were 38% in exsanguine Rhesus monkey at 8 hours after transfusion, and during that time, the hemoglobin and hematocrit of Rhesus monkey were maintained at the same level as before it lost blood. It is concluded that the modified pRBC can be safely transfused into Rhesus monkey and relieve the anemic symptom exsanguine Rhesus monkey. It suggested that pRBC can be hopefully used as a blood substitute for primate and human in the future.

[Evaluation of the glucose screening retest during pregnancy].

OBJECTIVE: To evaluate of the glucose screening retest for gestational diabetes mellitus (GDM) during pregnancy. METHODS: A retrospective analysis of 714 pregnant women screened for GDM, between December 1, 2001, and December 31, 2002, was performed. The first glucose challenge test (GCT) was performed in 16 - 27 week and retested in 28 - 38 week. Diagnosis of GDM was based on the criteria of Dong. NDDG criteria was also discussed. RESULTS: (1) 1-hour glucose value of 50 g GCT >or= 7.8 mmol/L was set as abnormal. The first 50 g GCT abnormal rate was 26.6% (190/714), and the retest abnormal rate was 35.2% (225/639). The mean age of pregnant women in 50 g GCT positive group was significantly higher than that in the negative group (P < 0.05), but there was no significant difference in family history and body mass index (BMI) between the two groups. Both the mean birth weight and the incidence of macrosomia of second 50 g GCT abnormal group were significantly higher than those in the normal group (P < 0.05). (2) By the criteria of Dong, 28 women were found to have GDM and 40 have IGT (impaired glucose tolerance) by the first 50 g GCT. New added cases included 15 GDM and 27 IGT by the retest 50 g GCT. By NDDG criteria, 14 GDM and 24 IGT cases were diagnosed by the first 50 g GCT, 5 GDM and 14 IGT cases by retest GCT. (3) The 1-hour blood glucose value [(7.3 +/- 1.6) mmol/L] in second 50 g GCT were significantly higher than those in first 50 g GCT [(6.9 +/- 1.8) mmol/L]. The results of 50 g GCT of two times were consistent in 68.1% women (normal/normal and abnormal/abnormal). There were 376 (52.7%) women whose 1-hour glucose value of the first 50 g GCT or= 7.8 mmol/L, and the 75 g OGTT should be done directly. (3) Maternal age appears to be the most important risk factor for GDM.

Establishment of a highly sensitive leptin radioimmunoassay and detection of increased leptin levels in hyperlipidemia and pregnancy.

The highly effective antibody has been obtained by immunizing rabbits with recombinant leptin many times. The leptin is iodinated with the chloramine-T method and purified with a Sephadex-G25 chromatography column. The reaction between antigen and antibody is carried out by a one-step balance method and cultured at 4 degrees C for 24 h; the binding and free antigen was then separated by PR reagent. The determining range of this method is about 0.5-24 ng/mL; limited detection level is 0.45 ng/mL, relative standard deviation in a group, and among groups, are less than 5.4% and 8%, respectively. The level of blood leptin in 277 samples of normal persons, in 112 samples of overweight persons (weight/hieght m2 > or = 25) and 224 samples of hyperlipidemic patients have been measured by this method. It is demonstrated that the level of blood leptin in males is much lower than that of the females, and becomes elevated with increased age. Serum leptin level in overweight persons and hyperlipidemic patients is also much higher than that of normal groups (P < 0.01). Serum leptin of 21 workers in our lab at 8:00 AM and 4:00 PM has been tested. It was found that there are no differences between the two time points. The same results are obtained within age groups. Leptin levels of pregnant women's serum is higher than those of the control group (P < 0.001). Leptin in newborn's serum is significantly lower than those of mothers (P < 0.01). There is no obvious correlation between leptin level of mother and newborns by correlation analysis (r = 0.19, P > 0.05). The body weight and body weight index of pregnant women are well correlated with their serum leptin levels (r = 0.33 and 0.35, P < 0.05). The body weight and body weight index of newborns are well correlated with their serum leptin levels (r = 0.54 and 0.49, P < 0.001). The serum leptin level of pregnant women is not correlated with newborn's body weight (r = 0.10). These results have shown that the proposed method is stable, simple, and specific, being sensitive enough to determine leptin levels in human serum or plasma.