High-throughput primer design by scoring in piecewise logistic model for multiple polymerase chain reaction variants.

Polymerase chain reaction (PCR) variants requiring specific primer types are widely used in various PCR experiments, including generic PCR, inverse PCR, anchored PCR, and ARMS PCR. Few tools can be adapted for multiple PCR variants, and many tools select primers by filtration based on the given parameters, which result in frequent design failures. Here we introduce PrimerScore2, a robust high-throughput primer design tool that can design primers in one click for multiple PCR variants. It scores primers using a piecewise logistic model and the highest-scored primers are selected avoiding the issue of design failure and the necessity to loosen parameters to redesign, and it creatively evaluates specificity by predicting the efficiencies of all target/non-target products. To assess the prediction accuracy of the scores and efficiencies, two next generation sequencing (NGS) libraries were constructed-a 12-plex and a 57-plex-and the results showed that 17 out of 19 (89.5%) low-scoring pairs had a poor depth, 18 out of 19 (94.7%) high-scoring pairs had a high depth, and the depth ratios of the products were linearly correlated with the predicted efficiencies with a slope of 1.025 and a coefficient of determination (R2) 0.935. 116-plex and 114-plex anchored PCR panels designed by PrimerScore2 were applied to 26 maternal plasma samples with male fetuses, the results showed that the predicted fetal DNA fractions were concordant with fractions measured in gold standard method (Y fractions). PrimerScore2 was also used to design 77 monoplex Sanger sequencing primers, the sequencing results indicated that all the primers were effective.

Phelan-McDermid Syndrome in Pediatric Patients With Novel Mutations: Genetic and Phenotypic Analyses.

Background: PhelanrMcDermid syndrome (PMS) is an uncommon autosomal dominant inherited developmental disorder. The main characteristics are hypotonia, intellectual disability, autism spectrum disorder, autism-like behaviors and tiny facial deformities. Most cases are caused by the deletion of the 22q13 genomic region, including the deletion of SHANK3. Methods: Genetic and phenotype evaluations of ten Chinese pediatric patients were performed. The clinical phenotypes and genetic testing results were collected statistically. We analyzed the deletion of the 22q13 genomic region and small mutations in SHANK3 (GRCh37/hg19) and performed parental genotype verification to determine whether it was related to the parents or was a novel mutation. Results: The age of the patients diagnosed with PMS ranged from 0 to 12 years old. Nine of the pediatric patients experienced Intellectual Disability, language motion development delay and hypotonia as prominent clinical features. One subject had autism, two subjects had abnormal electroencephalogram discharge and one subject was aborted after fetal diagnosis. Three patients had a SHANK3 mutation or deletion. All but the aborted fetuses had intellectual disability. Among the ten patients, a deletion in the 22q13 region occurred in seven patients, with the smallest being 60.6 kb and the largest being >5.5 Mb. Three patients had heterozygous mutations in the SHANK3 gene. Conclusion: All ten patients had novel mutations, and three of these were missense or frameshift mutations. For the first time reported, it is predicted that the amino acid termination code may appear before protein synthesis. The novel mutations we discovered provide a reference for clinical research and the diagnosis of PMS.

Case Report: Genetic Analysis of a Small Supernumerary Marker Chromosome in a Unique Case of Mosaic Turner Syndrome.

BACKGROUND: The aim of this study was to explore the source and morphology of a small supernumerary marker chromosome (sSMC) from karyotype analysis of a patient with a unique case of mosaic Turner syndrome. The study findings will provide technical reference and genetic counseling for similar cases. CASE PRESENTATION: A female patient with 46,X,+mar karyotype was diagnosed by genetic karyotype analysis. Genetic methods including fluorescence in situ hybridization (FISH) and copy number variation sequencing (CNV-seq) based on low-depth whole-genome sequencing were used to explore the source and morphology of sSMC. FISH technology showed that 56.5% of the cells were X and 43.5% of the cells were XY. CNV-seq detection found that the sSMC was chrY, implying that the patient's karyotype was mos 45,X[58.6%]/46,XY[41.4%]. Retrospective karyotype analysis indicated that the female patient's sSMC was inherited from her father's small chrY. Customized FISH probe of Yq12 microdeletion was positive, indicating that the sSMC was a del(Y)(q12). Based on the results of genetic diagnosis, the specialist doctor gave a comprehensive genetic consultation and ordered regular follow-up examinations. CONCLUSIONS: The findings of the current study showed that the chromosome description of the unique Turner case was mos 45,X[56.5%]/46,X,del(Y)(q12)[43.5%]. FISH technology played a key role in diagnosis of mosaicism. The terminal deletion of mosaic chrY provided a scientific and an accurate explanation for masculinity failure and abnormal sexual development of the current case.

Case Report: A Novel Compound Heterozygous Mutation of the FRMD4A Gene Identified in a Chinese Family With Global Developmental Delay, Intellectual Disability, and Ataxia.

Background: FERM domain-containing protein 4A (FRMD4A) is a scaffolding protein previously proposed to be critical in the regulation of cell polarity in neurons and implicated in human intellectual development. Case Presentation: We report a case of a 3-year-old boy with corpus callosum anomaly, relative macrocephaly, ataxia, and unexplained global developmental delay. Here, compound heterozygous missense mutations in the FRMD4A gene [c.1830G>A, p.(Met610Ile) and c.2973G>C, p.(Gln991His)] were identified in the proband, and subsequent familial segregation showed that each parent had transmitted a mutation. Conclusions: Our results have confirmed the associations of mutations in the FRMD4A gene with intellectual development and indicated that for patients with unexplained global developmental delay, the FRMD4A gene should be included in the analysis of whole exome sequencing data, which can contribute to the identification of more patients affected by this severe phenotypic spectrum.

Integrated miRNA-mRNA Expression Profiles Revealing Key Molecules in Ovarian Cancer Based on Bioinformatics Analysis.

Ovarian cancer is one of the leading causes of gynecological malignancy-related deaths. The underlying molecular development mechanism has however not been elucidated. In this study, we used bioinformatics to reveal critical molecular and biological processes associated with ovarian cancer. The microarray datasets of miRNA and mRNA expression profiles were downloaded from the Gene Expression Omnibus (GEO) database. Besides, we performed target prediction of the identified differentially expressed miRNAs. The overlapped differentially expressed genes (DEGs) were obtained combined with miRNA targets predicted and the DEGs identified from the mRNA dataset. The Cytoscape software was used to design a regulatory network of miRNA-gene. Moreover, the overlapped DEGs in the network were subjected to enrichment analysis to explore the associated biological processes. The molecular protein-protein interaction (PPI) network was used to identify the key genes among the DEGs of prognostic value for ovarian cancer, and the genes were evaluated via Kaplan-Meier curve analysis. A total of 186 overlapped DEGs were identified. Through miRNA-gene network analysis, we found that miR-195-5p, miR-424-5p, and miR-497-5p highly exhibited targeted association with overlapped DEGs. The three miRNAs are critical in the regulatory network and act as tumor suppressors. The overlapped DEGs were mainly associated with protein metabolism, histogenesis, and development of the reproductive system and ocular tissues. The PPI network identified 10 vital genes that promote tumor progression. Survival analysis found that CEP55 and CCNE1 may be associated with the prognosis of ovarian cancer. These findings provide insights to understand the pathogenesis of ovarian cancer and suggest new candidate biomarkers for early screening of ovarian cancer.

[Noninvasive prenatal genetic testing in 6804 pregnant women aged less than 35 years with positive results in serum screening].

OBJECTIVE: To investigate the feasibility of noninvasive prenatal genetic testing for detecting chromosome aneuploid in pregnant women aged less than 35 years with positive results in serum screening. METHODS: We analyzed the plasma cellfree fetal DNA in a total of 6804 pregnant women aged less than 35 years with singleton pregnancy from Foshan maternal and child health care hospital, whose weeks of gestation ranged from 12 to 24 weeks with ages on the expected date of confinement of 21-34 years. According to the results of serum screening, the women were divided to high-risk group and critical-risk group. Amniocentesis or cordocentesis was carried out if the results of noninvasive prenatal genetic testing were positive, and karyotyping or/and high-throughput sequencing was performed as the golden standard. All the women were followed up by telephone calls to assess the accuracy of the prenatal testing. RESULTS: Noninvasive prenatal testing was successfully completed in all the 6081 cases. In the high-risk group, 70 women with positive results were tested by noninvasive prenatal testing, among whom 53 were confirmed by karyotyping or high-throughput sequencing. In this group, the sensitivity, specificity and positive predictive value of trisomy 21 syndrome detection was 95.65%, 99.91% and 88.0%, respectively, with a false positive rate of 0.09% and a false negative rate of 4.35%; the sensitivity, specificity and positive predictive value for trisomy 18 syndrome was 100%, 100% and 100%, respectively, with false positive and false negative rates of 0; the false positive rate and false negative rate for trisomy 13 syndrome was 0.09% and 0, respectively; the sensitivity, specificity and positive predictive value for sex chromosome aneuploid as 100%, 99.80% and 30.0%, respectively, with a false positive rate of 0.2% and a false negative rate of 0; the sensitivity, specificity and positive predictive value for other chromosome aneuploid was 100%, 99.88% and 16.60%, respectively, with a false positive rate of 0.18% and a false negative rate of 0. In the critical risk group, 54 women with positive results received noninvasive prenatal genetic testing, among whom 36 were confirmed by karyotyping or high-throughput sequencing. The sensitivity, specificity and positive predictive value for trisomy 21 syndrome were all 100% and the false positive rate and false negative rate were both 0; the false positive rate was 0.11% and the false negative rate was 0 for trisomy 18 syndrome; the false positive rate and false negative rate for trisomy 13 syndrome was 0.04% and 0, respectively; the sensitivity, specificity and positive predictive value for sex chromosome aneuploid was 100%, 99.79% and 50.0%, respectively, with a false positive rate of 0.21% and a false negative rate of 0; the false positive rate for other chromosome aneuploid was 0.18% and the false negative rate was 0. No significant differences were found between the two groups in the sensitivity, specificity, positive predictive value and false positive rate for detection of trisomy 21 syndrome and sex chromosome aneuploid (P>0.05). CONCLUSIONS: Noninvasive prenatal genetic testing is necessary for high-risk pregnant women with critical-risk in serum screening who refuse invasive prenatal diagnosis, and it is highly sensitive and specific fir detecting chromosome aneuploid with low false positive and false negative rates.

A Cell-free DNA Barcode-Enabled Single-Molecule Test for Noninvasive Prenatal Diagnosis of Monogenic Disorders: Application to ß-Thalassemia.

Noninvasive prenatal testing of common aneuploidies has become routine over the past decade, but testing of monogenic disorders remains a challenge in clinical implementation. Most recent studies have inherent limitations, such as complicated procedures, a lack of versatility, and the need for prior knowledge of parental genotypes or haplotypes. To overcome these limitations, a robust and versatile next-generation sequencing-based cell-free DNA (cfDNA) allelic molecule counting system termed cfDNA barcode-enabled single-molecule test (cfBEST) is developed for the noninvasive prenatal diagnosis (NIPD) of monogenic disorders. The accuracy of cfBEST is found to be comparable to that of droplet digital polymerase chain reaction (ddPCR) in detecting low-abundance mutations in cfDNA. The analytical validity of cfBEST is evidenced by a ß-thalassemia assay, in which a blind validation study of 143 at-risk pregnancies reveals a sensitivity of 99.19% and a specificity of 99.92% on allele detection. Because the validated cfBEST method can be used to detect maternal-fetal genotype combinations in cfDNA precisely and quantitatively, it holds the potential for the NIPD of human monogenic disorders.

[The Design, Synthesis, Characteristics and Photoelectric Properties Research of A Bulky Steric Hindrance 9-Phenyl-Fluorene Functionalized Aniline Intermediate].

In this paper, with sulfuric acid as a catalyst and acetic acid as solution at the temperature of 120 ℃ and refluxing period for 24 hours, a sterically hindered bulky 9,9-diarylfluorene intermediate of [9-(4-anilino)-9-phenyl-fluorene] had been successfully designed and synthesized with Friedel-Crafts reaction. The molecular structure of this compound was characterized in detail with nuclear magnetic resonance hydrogen spectrum, mass spectrometry, infrared ray, and so on. Nuclear magnetic resonance hydrogen spectrum and infrared ray spectrum of the compound indicated that the characteristic functional group amino of the compound at 6.55 ppm, 3 481 and 3 385 cm-1, respectively. UV-visible and fluorescence spectra properties of 9-(4-anilino)-9-phenyl-fluorene were characterized and discussed preliminarily, respectively. The research results show that the 9-(4-anilino)-9-phenyl-fluorene with bulky steric hindrance effects has four main absorption peaks with wavelength of 243, 257, 298 and 311 nm in dichloromethane solution, respectively. Moreover, with the excitation wavelength of 308 nm, an emission spectrum curve was obtained with wavelength of 300～500 nm, which has the maximum emission peak of 328 nm with a slim peak at about 405 nm and a long tail to 500 nm. The long tail was probably attributed to the interaction of intermolecular hydrogen bonding from aniline. The appropriate scope of fluorescence emission (300~500 nm) make the compound overlap with the absorption spectra of the classic blue material Bis(3,5-difluoro-2-(2-pyridyl)phenyl-(2-carboxypyridyl)iridium (Ⅲ) (FIrpic) (300~500 nm). It is possible to obtain excellent host materials through proper molecular tailoring and prepare well for high performance light-emitting device. In order to further understand the photoelectric properties of the compound, we used acetonitrile as solvent and tetrabutylammonium hexafluorophosphate as electrolyte, and the electrochemical properties of the compound was characterized with cyclic voltammetry measurements.The onset of the reduction and oxidation potential of the compound are -0.759 and 0.898 V, and the corresponding HOMO and LUMO energy levels are -5.38 and -3.72 eV, which wolud be beneficial to holes and electrons injection/transportation and further modified to be excellent host materials. All of these data would provide a useful reference for further fabrication of organic semiconductor luminescent device with high performance.

Hyperbranched lysine-arginine copolymer for gene delivery.

Based on the reactivity of amine groups and carboxyl groups of L-lysine and L-arginine, thermal polymerization of these two natural amino acids results in hyperbranched lysine-arginine copolymers (P-lys-argX, where X refers to the relevant molar ratio of arginine to lysine). Hyperbranched polylysine (P-lys) and two derivatives (P-lys-arg0.10 and P-lys-arg0.20) have been prepared. The arginine-rich hyperbranched polymers can interact with plasmid DNA to form nano-sized particles. The polyplexes were physicochemically analyzed by agarose gel electrophoresis, dynamic light scattering, and zeta potential measurements. Furthermore, their transfection efficiency was assessed, employing COS-7, 293T, and HeLa cell lines. It was found that P-lys showed poorly in its ability of condensation with DNA and transfection efficiency. On the other hand, arginine-rich products resulted to significant enhancement of its transfection efficiency, which is dependent on the content of arginine in the polymers, and the cell line used. P-lys-arg0.20 exhibited better transfection efficiency under all the condition studied. Besides, P-lys-arg0.20 showed lower toxicity in COS-7 cells.

Hydrothermal preparation and electrochemical sensing properties of TiO(2)-graphene nanocomposite.

A facile hydrothermal method has been developed and shown to be effective for the preparation of TiO(2)-graphene nanocomposite. The as-prepared nanocomposite was characterized using FT-IR spectroscopy, powder X-ray diffraction (XRD) and scanning electron microscopy (SEM). The TiO(2)-graphene modified glassy carbon electrode (GCE) exhibited remarkable electron transfer kinetics and electrocatalytic activity toward the oxidation of dopamine (DA). Furthermore, the oxidation of common interfering agent such as ascorbic acid (AA) was significantly suppressed at this modified electrode, which resulted in good selectivity and sensitivity for electrochemical sensing of DA. These results demonstrate that the TiO(2)-graphene hybrid material has promising potential applications in electrochemical sensors and biosensors design.