Application of Restriction Site-Associated DNA Sequencing (RAD-Seq) for Copy Number Variation and Triploidy Detection in Human.

At present, low-pass whole-genome sequencing (WGS) is frequently used in clinical research and in the screening of copy number variations (CNVs). However, there are still some challenges in the detection of triploids. Restriction site-associated DNA sequencing (RAD-Seq) technology is a reduced-representation genome sequencing technology developed based on next-generation sequencing. Here, we verified whether RAD-Seq could be employed to detect CNVs and triploids. In this study, genomic DNA of 11 samples was extracted employing a routine method and used to build libraries. Five cell lines of known karyotypes and 6 triploid abortion tissue samples were included for RAD-Seq testing. The triploid samples were confirmed by STR analysis and also tested by low-pass WGS. The accuracy and efficiency of detecting CNVs and triploids by RAD-Seq were then assessed, compared with low-pass WGS. In our results, RAD-Seq detected 11 out of 11 (100%) chromosomal abnormalities, including 4 deletions and 1 aneuploidy in the purchased cell lines and all triploid samples. By contrast, these triploids were missed by low-pass WGS. Furthermore, RAD-Seq showed a higher resolution and more accurate allele frequency in the detection of triploids than low-pass WGS. Our study shows that, compared with low-pass WGS, RAD-Seq has relatively higher accuracy in CNV detection at a similar cost and is capable of identifying triploids. Therefore, the application of this technique in medical genetics has a significant potential value.

Hepatic Interferon Regulatory Factor 6 Alleviates Liver Steatosis and Metabolic Disorder by Transcriptionally Suppressing Peroxisome Proliferator-Activated Receptor γ in Mice.

Nonalcoholic fatty liver disease (NAFLD) has become a worldwide epidemic. A large and growing unmet therapeutic need has inspired numerous studies in the field. Integrating the published genomic data available in the Gene Expression Omnibus (GEO) with NAFLD samples from rodents, we discovered that interferon regulatory factor 6 (IRF6) is significantly downregulated in high-fat diet (HFD)-induced fatty liver. In the current study, we identified IRF6 in hepatocytes as a protective factor in liver steatosis (LS). During HFD challenge, hepatic Irf6 was suppressed by promoter hypermethylation. Severity of HFD-induced LS was exacerbated in hepatocyte-specific Irf6 knockout mice, whereas hepatocyte-specific transgenic mice overexpressing Irf6 (IRF6-HTG) exhibited alleviated steatosis and metabolic disorder in response to HFD feeding. Mechanistic studies in vitro demonstrated that hepatocyte IRF6 directly binds to the promoter of the peroxisome proliferator-activated receptor γ (PPARγ) gene and subsequently halts the transcription of Pparγ and its target genes (e.g., genes that regulate lipogenesis and lipid acid uptake) under physiological conditions. Conclusion: Irf6 is downregulated by promoter hypermethylation upon metabolic stimulus exposure, which fail to inhibit Pparγ and its targets, driving abnormalities of lipid metabolism.

[Alleles dropout patterns of Y-short tandem repeats in infertile males with Y chromosome microdeletions].

Y chromosomal short tandem repeat (Y-STR) typing is the most commonly used genetic technique in forensic studies. However, there may be a limit to the application of Y-STR in forensic science as Y-STR loci are subject to loss or variation caused by the higher chromosomal structures' spontaneous mutation rate. Located in the long arm of the Y chromosome, azoospermia factor (AZF) have been shown to participate in spermatogenesis and its deletion could cause infertility. However, little is known about the Y-STR dropout pattern in individuals with Y chromosome microdeletions. In this study, 85 infertile males with Y chromosome interstitial deletion were identified and special Y-STR allele dropout patterns were analyzed by employing a Y-STR Commercial Kit and a Y chromosome Deletion Kit. Results demonstrate that AZF a region deletion are related to DYS439-DYS389I-DYS389II alleles dropout, while AZF b region or c region deletions correlate to DYS448 allele dropout. Null DYS385-DYS392-DYS448 alleles were observed in AZF b+c+d region deletion individuals. While null DYS390-Y-GATA-H4-DYS385-DYS392-DYS448 alleles were observed in AZF a+b+c+d large region deletion individuals. Our data suggest that Y chromosome microdeletions may indicate specific Y-STR locus dropout patterns.

[Clinical value of MLPA in the prenatal gene diagnosis of Duchenne muscular dystrophy].

OBJECTIVE: To investigate the clinical value of multiplex ligation-dependent probe amplification (MLPA) in the prenatal gene diagnosis of high risk pregnant women from Duchenne muscular dystrophy (DMD) families. METHODS: The 155 high risk pregnant women from DMD families were recruited from 2005 to 2012 in 4 hospitals in Guangzhou, such as Southern Hospital of Southern Medical University and the Third Affiliated Hospital of Guangzhou Medical University. Among all the samples, 7 were chorionic villus samples taken from early-stage pregnancy and 148 were amniotic fluid samples from mid-stage pregnancy. After the maternal contamination was eliminated, the fetal DMD gene screening was carried out by using MLPA. The mutation rates in DMD exons were calculated in all the 155 families. RESULTS: (1) Among the 155 fetuses of the DMD high risk pregnant women, there were 72 male fetuses and 83 female fetuses. In the male fetuses, there were 27 sufferers (38%). In the female fetuses, there were 28 carriers (34%). And there were 100 normal fetuses. (2) Among the 27 DMD sufferers, 22 cases were DMD exon homozygous deletions (14.2%, 22/155) and 5 cases were DMD exon duplications (3.2%, 5/155). Among the 28 carriers, 25 cases were gene heterozygous deletions (16.1%, 25/155) and 3 cases were gene heterozygous duplications (1.9%, 3/155). In the 155 families, the DMD mutations mainly occurred in exons 45-52, and the exon 49 had the highest mutation rates of 22 times. (3) Among the 7 cases of prenatal gene diagnosis using chorionic villus samples, 2 fetuses had the identical DMD genotypes with their mothers and probands. One was a DMD sufferer and the other was a carrier. Termination or continuation of pregnancy was suggested based on the genotype of the fetus. CONCLUSIONS: MLPA provides an accurate method in the prenatal diagnosis of DMD. It could be used to distinguish DMD gene homozygous deletions from heterozygous deletions and duplications. Therefore, it is valuable for DMD prenatal diagnosis in high-risk women. Chorionic villus sampling can be applied to the early prenatal diagnosis for DMD disease.

[Establishment of an internal control for directed differentiation using pluripotent stem cell lines derived from heterozygotic twins].

OBJECTIVE: To reprogram amniotic fluid cells into pluripotent stem cells in order to create an optimal internal control model for directed cell differentiation. METHODS: Human amniotic fluid-derived cells (hAFDCs) from heterozygotic twin fetuses were induced by retroviral vectors encoding Oct4, Sox2, c-Myc and Klf4. In vivo pluripotency, differentiation capacity and karyotype of hAFDCs induced pluripotent stem cells (hAFDCs-iPSCs) were determined. RESULTS: hAFDC-iPSCs derived from heterozygotic twins have maintained self renewal, with expression of high pluripotency marker gene detected at both mRNA and protein levels. The cells have maintained their differentiation capacity both in vitro and vivo, and showed normal karyotypes after long-term culturing in vitro. CONCLUSION: hAFDCs-iPSCs derived from heterozygotic twins have good consistency in terms of genetic background, and can provide a good internal control for directed differentiation of iPSCs, and may be used an ideal source for autologous cell replacement therapy in the later life of the fetus.

[Analysis of de novo copy number variations in a family affected with autism spectrum disorders using high-resolution array-based comparative genomic hybridization].

OBJECTIVE: To analyze de novo copy number variations (CNVs) in a Chinese family affected with autism spectrum disorders (ASD). METHODS: Affymetrix Cytogenetics Whole Genome 2.7M Array assay was performed to identify potential CNVs in four members from the family. RESULTS: A total of 89 de novo CNV regions were identified in the autistic siblings. The CNV regions in total have exceeded 1/1000 of the lengths of chromosomes 5, 11 and 14. In addition, de novo CNV regions were also identified at 3p26.1, 4q22.2, and 5p15.2, which encompassed 10 genes associated with nerve development including GRM7, GRID2 and CTNND2. CONCLUSION: A number of nerve development associated genes were at the de novo CNV sites, which may provide new clues for genetic research of ASD. High-resolution array-comparative genomic hybridization is an effective method for detecting submicroscopic chromosomal imbalances.

Relationship Between Depression After Hemorrhagic Stroke and Auditory Event-Related Potentials in a Chinese Patient Group.

Purpose: Post-stroke depression (PSD) is the most common psychiatric sequelae of stroke. Numerous studies revealed that event-related potentials (ERP) can reflect depression severity to a certain extent, while there is almost no research on depression after hemorrhagic stroke. Therefore, we employed a prospective cross-sectional study to explore the relationship between ERP and depression after hemorrhagic stroke. Methods: A total of 74 patients with intracranial hemorrhage were included in this study. Neurological deficits were evaluated using the National Institutes of Health Stroke Scale (NIHSS) on admission. Depression severity and cognitive impairment were measured using the 17-item Hamilton Depression Scale (HAMD-17) and the Chinese version of the Montreal Cognitive Assessment (MoCA) after two weeks of treatment. All patients were conducted auditory Oddball paradigm for event-related potential mismatch negativity (MMN) and P300. Results: In total, 36 patients were diagnosed with PSD at the two weeks of treatment, for a percentage of 48.6%. Depression severity of ICH patients correlated positively with both the latency of MMN (r = 0.376, P = 0.001) and P300 (r = 0.325, P = 0.005), and correlated negatively with both the amplitude of MMN (r=-0.385, P = 0.001) and P300 (r=-0.311, P = 0.007). Depression severity was negatively correlated with cognitive function after hemorrhagic stroke (r=-0.347, P = 0.002). Conclusion: The latency and amplitude of MMN and P300 can well reflect the degree of depression after hemorrhagic stroke, which may help in the early diagnosis and effective treatment of PSD.

Hypomethylated and hypermethylated profiles of H19DMR are associated with the aberrant imprinting of IGF2 and H19 in human hepatocellular carcinoma.

In this study, 39 human hepatocellular carcinoma (HCC) tissues and 7 normal adult liver tissues were screened for heterozygous polymorphisms in IGF2, H19, and the differentially methylated region of H19 (H19DMR) using PCR-RFLP and PCR sequencing. The imprinting of IGF2 and H19 was examined by RT-PCR-RFLP, while the methylation profile of H19DMR was detected by bisulfite sequencing from every informative sample. Of the informative HCC samples 47.06% (8 of 17) demonstrated a gain of imprinting of IGF2, and 21.74% (5 of 23) of the informative HCC samples demonstrated a loss of imprinting of H19. Interestingly, we found three methylation profiles for H19DMR in the informative HCC samples: hyper-, medium-, and hypomethylated profiles. Furthermore, the hypomethylated and hypermethylated profiles were immediately associated with aberrant imprinting of IGF2 and H19.

[Separation and purification and structural characterization of baicalein].

The root of scutellaria baicalensis georgi that contains a variety of flavonoids is a very old and well-known drug in traditional Chinese medicine, which is widely used for treatment of bronchitis, tumors and inflammatory diseases. The baicalein is the main active component from traditional Chinese medicine-scutellaria baicalensis georgi. It is a very significance research work that the baicalein was separated and purified, and its composition and molecular structure are analyzed and determined for the pharmacology study of Chinese medicine-scutellaria baicalensis georgi. The main works in this paper are as follows. Powdered roots (100 g) were extracted with methanol by three times, each time for 48 hours. The crude extracts were purified by polyamide column chromatography and CH3Cl-C2H5OH gradient desorption. A short yellow prismatic crystal was acquired by recrystallizing technique and its composition and molecular structure were characterized by color reactions and spectral analysis methods as FTIR, UV-Vis, MS and 1H NMR, 13C-NMR. The FTIR spectrum appears the absorption bands for hydroxyls, pyrone carbonyl, aromatic C=C bond and singly substituted phenyl. The characteristic absorption peaks and the vibration modes in FTIR spectrum were identified as corresponding groups. The UV-Vis spectrum in methanol solution and the mix solution of methanol with 5 diagnostic reagents, NaOMe, NaOAc, NaOAc/H3BO3, AlCl3, AlCl3/HCl, respectively indicate that the yellow prismatic crystal is flavone with 5-hydroxyl, 4-carbonyl and 5,6,7- or 5,7,8-trihydroxyls on ring A. The structure of the crystal was characterized by three different MS. The results of FAB-, ESI- and EI-MS show that it is not a flavone glocuside but the flavone with three phenyl hydroxyls on ring A, and no OH group and other substituted groups on ring B. The molecular ion and fragment ions are identified by MS, which include such as m/z 270 M+, m/z 242 [M-CO]+, m/z 168 A, m/z 140 [A1-CO]+, m/z 105 B, m/z 102 B, m/z 77 [B2-CO]+, respectively. 13C-NMR (DMSO-d6) exhibits the signals of the fifteen carbon atoms, nine oxygenous aromatic C, five non-oxygenous aromatic C and a carbonyl C. 1H-NMR(DMSO-d6 + D2O, DMSO-d6 )indicates the presence of C-5, C-6, C-7 hydroxyl protons, which is consistent with the results of UV spectrum. The signals for C-2',6' hydroxyls appear at delta = 8.055 as a doublet peak with spin-spin coupling constant 6.0 Hz. The other signals were ascribed to the corresponding H or C atoms in the compound. The results of FTIR, UV-Vis, MS, 1H NMR, 13C-NMR spectroscopy characterization show that crystal is the 5,6,7-trihydroxy-flavone, that is baicalein, and the molecular formula is C15H10O5.

[UV-Vis spectroscopic characterization of active components from Scutellaria baicalensis georgi].

The molecular structures of three active components from scutellaria baicalensis have been studied by ultraviolet-visible spectra. The index flavonoid structures and substituted positions were deduced by analyzing the UV-Vis spectra in the methanol solution of three active components and the methanol solution with 5 diagnostic reagents, NaOMe, NaOAc, NaOAc/H3BO3, AlCl3, AlCl3/HCl respectively, which provided strong evidences for the structural characterization of the active components from natural products.

[The aberrant imprinting of insulin-like growth factor II and H19 in human hepatocellular carcinoma].

OBJECTIVE: To investigate the allelic expression of IGF2/H19 in human hepatocellular carcinoma and to analyze the relationship between the imprint status of IGF2 and that of H19. METHODS: Heterozygotes of IGF2 and of H19 were identified by restriction fragment length polymorphism (RFLP). Allelic expression was detected by reverse transcription-polymerase chain reaction (RT-PCR) and RFLP. RESULTS: Forty seven point one percent (47.1%, 8 of 17) of HCC samples were demonstrated to have the gain of imprinting (GOI) of in IGF2 gene, and 45.5% (5 of 11) of HCC samples were found to have the loss of imprinting (LOI) for H19 gene. No relationship was observed between the imprinting status of IGF2 and that of H19. CONCLUSIONS: IGF2 GOI and H19 LOI are common in HCC, and the imprinting of IGF2 could be independent from that of H19 in adult liver.

[The imprinting status and expression of insulin-like growth factor 2 gene in human hepatocellular carcinoma].

OBJECTIVE: To study the imprinting status and expression level of insulin-like growth factor 2 (IGF2) gene in hepatocellular carcinoma and to provide a clue for elucidating the mechanism of carcinogenesis of hepatocellular carcinoma. METHODS: The heterozygote status of IGF2 gene was detected by restriction fragment length polymorphism. The imprinting status and expression level of IGF2 were evaluated by semi-quantitative reverse transcription-polymerase chain reaction. RESULTS: It was found that 10/18 (55.6%) of hepatocellular carcinoma showed the gain of imprinting (GOI), with 6/10 (60%) adjacent cirrhosis of liver tissues also displaying GOI of IGF2. Overexpression of IGF2 in cancer tissues was detected in 9/18 (50%) samples, but no significant difference was observed among each imprinting status. CONCLUSION: GOI of IGF2 may take part in human hepatocellular carcinogenesis.