Identification of key genes and pathways of BMP-9-induced osteogenic differentiation of mesenchymal stem cells by integrated bioinformatics analysis.

BACKGROUND: The purpose of present study was to identify the differentially expressed genes (DEGs) associated with BMP-9-induced osteogenic differentiation of mesenchymal stem cells (MSCs) by using bioinformatics methods. METHODS: Gene expression profiles of BMP-9-induced MSCs were compared between with GFP-induced MSCs and BMP-9-induced MSCs. GSE48882 containing two groups of gene expression profiles, 3 GFP-induced MSC samples and 3 from BMP-9-induced MSCs, was downloaded from the Gene Expression Omnibus (GEO) database. Then, DEGs were clustered based on functions and signaling pathways with significant enrichment analysis. Pathway enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) demonstrated that the identified DEGs were potentially involved in cytoplasm, nucleus, and extracellular exosome signaling pathway. RESULTS: A total of 1967 DEGs (1029 upregulated and 938 downregulated) were identified from GSE48882 datasets. R/Bioconductor package limma was used to identify the DEGs. Further analysis revealed that there were 35 common DEGs observed between the samples. GO function and KEGG pathway enrichment analysis, among which endoplasmic reticulum, protein export, RNA transport, and apoptosis was the most significant dysregulated pathway. The result of protein-protein interaction (PPI) network modules demonstrated that the Hspa5, P4hb, Sec61a1, Smarca2, Pdia3, Dnajc3, Hyou1, Smad7, Derl1, and Surf4 were the high-degree hub nodes. CONCLUSION: Taken above, using integrated bioinformatical analysis, we have identified DEGs candidate genes and pathways in BMP-9 induced MSCs, which could improve our understanding of the key genes and pathways for BMP-9-induced osteogenic of MSCs.

[Establishment of an allele-specific PCR method for direct screening of CYP21A2 gene mutation].

OBJECTIVE: To establish an allele-specific PCR method for detect screening of CYP21A2 gene mutation. METHODS: Allele-specific PCR primers and analogy primers were designed based on the sequence alignment of CYP21A2 and CYP21AP genes. Genomic DNA was extracted from blood specimens of 4 patients with 21-hydroxylase deficiency and 5 healthy controls and respectively amplified with allele-specific PCR primers and analogy primers and sequenced. RESULTS: Mutations of CYP21A2 including IVS2-13A/C>G, Arg356Trp and Arg149Pro were found with the established method in all of the 4 patients but not in the healthy controls. When detected with the analogy primers set, IVS2-13A/C>G and Arg356Trp were observed in both patients and healthy controls. CONCLUSION: The allele-specific PCR-based method is a simple, effective and reliable method for the detection of CYP21A2 gene mutation.

[Application of denaturing high performance liquid chromatography for the detection of maternal DNA contamination during prenatal diagnosis].

OBJECTIVE: To establish a high-quality method for detecting short tandem repeats(STR) using denaturing high performance liquid chromatography(DHPLC) in order to exclude maternal contamination and improve the accuracy of prenatal diagnosis. METHODS: Two families were recruited. DNA was extracted from blood samples from the parents as well as amniotic fluid. Sixteen STR sites were amplified and analyzed based on the range of allele length reported by a STR database. Maternal DNA was mixed with DNA derived from amniotic fluid samples with the ratio 1:1, 1:4, 1:9, 1:19 and 1:99. vWA STR site was detected with DHPLC to confirm the sensitivity of detection. RESULTS: Sixteen STR sites were analyzed by DHPLC, for which at least 10 were found to be different between the mothers and fetuses. The detection rate, with maternal contamination excluded, was 66.7%. And the sensitivity of detection was 1-10%. CONCLUSION: Maternal contamination of amniotic fluid can be rapidly excluded with accuracy with DHPLC, which features a high sensitivity and good quality control, and can meet the European standards and provide a reliable quality control platform for prenatal diagnosis.

[Mutation analysis and prenatal diagnosis of FBN1 gene mutations for four patients with Marfan syndrome].

OBJECTIVE: To screen for mutations of fibrillin-1 (FBN1) gene in 4 patients with Marfan syndrome in order to provide prenatal diagnosis and genetic counseling. METHODS: Potential mutations of the FBN1 gene in the probands were detected with PCR and DNA sequencing. Subsequently, genomic DNA was extracted from amniotic fluid sampled between 18 to 20 weeks gestation. The mutations were confirmed with denaturing high-performance liquid chromatography - robust microsatellite instability (DHPLC-MSI) analysis with maternal DNA as reference. The products were further analyzed by direct sequencing and BLAST search of NCBI database. RESULTS: An IVS46+1G>A substitution was identified in patient A at +1 position of intron 46 of the FBN1 gene. Two novel missense mutations were respectively discovered at positions +4453 of intron 35 in patient B (Cys1485Gly) and position +2585 of intron 21 in patient C (Cys862Tyr). In patient D, a novel deletion (c.3536 delA) was found at position +3536 of intron 28. In all of the 4 cases, the same mutations have been identified in the fetuses. CONCLUSION: FBN1 gene analysis can provide accurate diagnosis of Marfan syndrome, which can facilitate both prenatal diagnosis and genetic counseling.

Dominant contribution of northern chinese to the paternal genetic structure of Chaoshanese in South China.

We investigated the Y chromosome of various Chinese populations to determine the patrilineal origin of the Chaoshanese population. Admixture analysis of six specific Y short tandem repeat (STR) loci in 6,292 individual samples taken from 51 populations, including Chaoshanese and Minnanese of our earlier studies, showed that over 85% of the Chaoshanese Y chromosomes were derived from the Central China Han (M (RH): 0.8614; M (BE): 1.1868 ± 0.2054), and a very small portion were from the southern aborigines. These results support a Central China Han origin of the Chaoshanese and additionally reveal that males from the Central China Han were the predominant contributor to the patrilineal genetics of the Chaoshanese. A phylogenetic tree and analysis of molecular variance signified a strong association between Y chromosomes of Chinese populations and their linguistic affiliations, revealing a coevolution of Y chromosome diversity and languages in East Asia.