AIM: To investigate the effect of homocysteine(Hcy) on the apoptosis of endothelial cells (EC). METHODS: First-passaged human umbilical vein endothelial cells (hUVEC) were cultured with M199 containing 3 mmol/L Hcy. hUVEC apoptosis was detected as follow: demonstration of nuclear changes by Hoechst 33258 staining, agarose gel electrophoresis of DNA fragments, detection of apoptotic cells by flow cytometry following Annexin V-PI doubled stain, Western blot for P53 and Bax protein detection and colorimetry detecting caspase-3 activity. RESULTS: Compared with control, homocysteine induced characteristic apoptotic changes in hUVEC. The chromosomal DNA of hUVEC appeared “DNA ladder” by agarose gel electrophoresis. Apoptotic cells were increased significantly (P

AIM: To investigate the influence of bone marrow stromal cells (BMSCs) on the anoxia cardiomyocyte apoptosis. METHODS: Using the anaerobic culture apparatus, the apoptosis of the cardiomyocytes, the BMSCs alone and co-cultured with each other were detected by morphological observation, PI staining flowcytometry, electrophoretic gel mobility analysis of DNA fragmentation. Western blotting was used to detect Bax and Bcl-2 protein expression. RESULTS: Compared with control, the BMSCs were unsensitive to anoxic cultured while the anoxic cardiomyocytes were prone to apoptosis. Apoptosis of cardiomyocytes was increased significantly, detected by PI staining and agarose gel elestrophoresis showed “DNA ladder”. However, when anoxia cardiomyocytes co-cultured with BMSCs, apoptosis cells were decreased, “DNA ladder” disappeared and the expression of protein Bax was also decreased. CONCLUSION: Bone marrow stromal cells prevent the anoxia cardiomyocytes from apoptosis, probably by suppressing the expression of Bax protein.

OBJECTIVETo explore the source of small supernumerary marker chromosome in a case.

METHODSG-banded karyotyping, fluorescence in situ hybridization, multiple sequence tagged sites (STS) of the Y chromosome, and Illumima Human Cyto SNP-12 Beadchip analysis were carried out.

RESULTSThe karyotype was mos 46,X,+mar1[21]/46,X,+mar2[78]. Y chromosome STS analysis has displayed the presence of sy84, sY86, USP9Y and DDX3Y genes from the AZFa region, and sY1227 of the AZFb region, while sY1228, sY1015, sY127, sY134 from the AZFb region, and sY254 and sY255 from the AZFc region were missing. FISH analysis has verified both of the marker chromosomes to be Y chromosome fragments. Mar1 was ish.idic(Y)(q11.2)(SRY++,DXZ1+,DYZ3++,DYZ1-), while mar2 was ish.del(Y)(q11.2)(SRY+,DXZ1+,DYZ3+,DYZ1-). Single nucleotide polymorphism (SNP) microarray analysis showed that the Yq11.2-Yq12 has lost a 10.81 Mb fragment.

CONCLUSIONThe marker chromosomes were verified to be aberrant Y chromosomes, with the breakage and recombination occurring in Yq11.2. Mar 1 was an isodicentric Y chromosome (idic(Y)pter to q11.2::q11.2 to pter), and mar2 was del(Y)(q11.2). The karyotype was mos 46,X,ish idic(Y)(q11.2)(DYZ3++,SRY++,DXZ1+,DYZ1-)[21]/46,X,ish del(Y)(q11.2)(DYZ3+,SRY+,DXZ1+,DYZ1-)[78]. Combined FISH, Y chromosome STS analysis, SNP microarray analysis and other technologies can facilitate determination of the nature of marker chromosomes.

Adult ; Chromosomes, Human, Y ; genetics ; Cytogenetics ; Humans ; In Situ Hybridization, Fluorescence ; Male ; Polymorphism, Single Nucleotide ; Sex Chromosome Aberrations ; Sex Chromosome Disorders ; genetics

OBJECTIVETo analyze the characteristics of azoospermia factor(AZF) deletions in Y-chromosome.

METHODSBased on the AZF microdeletion screening on 272 cases of azoospermia and 240 cases of severe oligozoo spermia, 49 cases were investigated using 23 sequence-tagged sites (STS) in AZFa, AZFb and AZFc. For some cases, single nucleotide rarians (SNV) method was applied to identify the single nucleotide polymorphism (SNPs) in four DAZ gene copies and to determine the copy number of the DAZ gene.

RESULTSIn 6 cases with deletions of AZFb+c, there was 1 case with sY98/sY1206 deletion, 4 cases with P5/distal-P1 recombination and 1 with P4/distal-P1 recombination. In 3 cases with deletions in AZFb, 1 case showed P5/P3 deletion and 2 cases showed P5/proximal-P1 recombination with DAZ1 and DAZ2 deletions. b2/b4 recombination was observed in all the 40 cases with deletions in AZFc. A fraction of patients with AZFb and AZFb+c deletions showed oligospermia and spermatogenic failure by testicular biopsy.

CONCLUSIONBreakpoint localization of deletions in AZF regions may help elucidating the mechanisms of microdeletions, and analysis of the characteristics and quantity of deleted genes essential for normal spermatogenesis may evaluate the association of phenotype with spermatogenic failure.

Chromosome Deletion ; Chromosomes, Human, Y ; genetics ; Deleted in Azoospermia 1 Protein ; Gene Dosage ; Genetic Loci ; Humans ; Male ; Oligospermia ; genetics ; physiopathology ; RNA-Binding Proteins ; genetics ; Seminal Plasma Proteins ; genetics ; Spermatogenesis

OBJECTIVETo analyze chromosome aberration in a child with mental retardation and abnormalities and its parents.

METHODSChromosome G banding, multiplex ligation-dependent probe amplification, fluorescence in situ hybridization and single nucleotide polymorphisms array were employed for analysis.

RESULTSKaryotype analysis revealed that the child was 46,XX and the father was 46,XY, while the mother was 46,XX, add (12)(p13). Subtelomeric region analysis with MLPA displayed that the child has reduced ACP1 gene copy number in 2p25 region and increased SLC6A12,KDM5A gene copy numbers in 12p11 region. SNP-array has fine mapped the duplication to 12p13.33-p12.3, a 15.142 Mb region, and a deletion to 2p25.3 for 3.194 Mb, which resulted in duplication of 9 genes including SLC6A12 as well as deletion of 11 genes including SNTG2, respectively. FISH analysis revealed that the child was 46,XX,ish,der(2),t(2;12)(p25;p13)mat, or partial monosomy 2p25 and partial trisomy 12p13. In addition,the mother was a carrier with cryptic balanced translocation chromosome, 46,XX,isht(2;12) (p25;p13). Mental abnormalities and retardation of the child may be attributed to heterozygous deletion of SNTG2, MYT1L genes and duplication of SLC6A12 gene.

CONCLUSIONCombined use of MLPA, FISH and SNP-array can facilitate accurate diagnosis of cryptic rearrangement at genomic level.

Adolescent ; Adult ; Carrier Proteins ; genetics ; Child ; Child, Preschool ; Chromosome Banding ; Chromosome Deletion ; Chromosomes, Human, Pair 12 ; genetics ; Chromosomes, Human, Pair 2 ; genetics ; Female ; Gene Rearrangement ; Humans ; Intellectual Disability ; diagnosis ; genetics ; Male ; Pedigree ; Protein Tyrosine Phosphatases ; genetics ; Proto-Oncogene Proteins ; genetics ; Translocation, Genetic ; Trisomy ; Young Adult

Background: No currently available biomarkers or treatment regimens fully meet therapeutic needs of type 1 diabetes mellitus (T1DM). Circular RNA (circRNA) is a recently identified class of stable noncoding RNA that have been documented as potential biomarkers for various diseases. Our objective was to identify and analyze plasma circRNAs altered in T1DM. Methods: We used microarray to screen differentially expressed plasma circRNAs in patients with new onset T1DM (n=3) and age-/gender-matched healthy controls (n=3). Then, we selected six candidates with highest fold-change and validated them by quantitative real-time polymerase chain reaction in independent human cohort samples (n=12). Bioinformatic tools were adopted to predict putative microRNAs (miRNAs) sponged by these validated circRNAs and their downstream messenger RNAs (mRNAs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to gain further insights into T1DM pathogenesis. Results: We identified 68 differentially expressed circRNAs, with 61 and seven being up- and downregulated respectively. Four of the six selected candidates were successfully validated. Curations of their predicted interacting miRNAs revealed critical roles in inflammation and pathogenesis of autoimmune disorders. Functional relations were visualized by a circRNA-miRNA-mRNA network. GO and KEGG analyses identified multiple inflammation-related processes that could be potentially associated with T1DM pathogenesis, including cytokine-cytokine receptor interaction, inflammatory mediator regulation of transient receptor potential channels and leukocyte activation involved in immune response. Conclusion Our study report, for the first time, a profile of differentially expressed plasma circRNAs in new onset T1DM. Further in silico annotations and bioinformatics analyses supported future application of circRNAs as novel biomarkers of T1DM.

No currently available biomarkers or treatment regimens fully meet therapeutic needs of type 1 diabetes mellitus (T1DM). Circular RNA (circRNA) is a recently identified class of stable noncoding RNA that have been documented as potential biomarkers for various diseases. Our objective was to identify and analyze plasma circRNAs altered in T1DM.

We used microarray to screen differentially expressed plasma circRNAs in patients with new onset T1DM (n=3) and age-/gender-matched healthy controls (n=3). Then, we selected six candidates with highest fold-change and validated them by quantitative real-time polymerase chain reaction in independent human cohort samples (n=12). Bioinformatic tools were adopted to predict putative microRNAs (miRNAs) sponged by these validated circRNAs and their downstream messenger RNAs (mRNAs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to gain further insights into T1DM pathogenesis.

We identified 68 differentially expressed circRNAs, with 61 and seven being up- and downregulated respectively. Four of the six selected candidates were successfully validated. Curations of their predicted interacting miRNAs revealed critical roles in inflammation and pathogenesis of autoimmune disorders. Functional relations were visualized by a circRNA-miRNA-mRNA network. GO and KEGG analyses identified multiple inflammation-related processes that could be potentially associated with T1DM pathogenesis, including cytokine-cytokine receptor interaction, inflammatory mediator regulation of transient receptor potential channels and leukocyte activation involved in immune response.

Our study report, for the first time, a profile of differentially expressed plasma circRNAs in new onset T1DM. Further in silico annotations and bioinformatics analyses supported future application of circRNAs as novel biomarkers of T1DM.

Objective To further investigate the expression of MAGE-1 gene in hepatocellular carcincma (HCC). Methods The tumors and adjacent liver tissue from 45 HCC patients and liver tissue from 28 non-HCC patients (16 with liver cirrhosis and 12 with normal liver) were characterized by RT-PCR. A 421 bp PCR product from a cDNA fragment spanning exons 1,2 and 3 was sequenced. The HLA type wes assayed by standard ELISA in 43 HCC patients. Results Thirty-two of 45 tumor tissues from HCC patients expressed MAGE-1 mRNA (71.1%).In contrast, MAGE-1 mRNA was not detected in adjacent tissues. Three were found to have point mutations at 3 idntical sites resulting in the substitution of two amino acid residues.The most frequent HLA types in 43 HCC patients were: HLA-A2, 53.5%; A11, 25.6%; A24, 20.9%; A33, 20.9%; HLA-B13, 28.3% and B35, 23.2%. Expression of HLA-A33 (20.9%) was higher in HCC patients than that predicted in the normal Chinese population (8.8%). There was no discemable correlation between MAGE-1 expression and α-FP level, tumor size and hepatitis B or C virus infection.The identification of peptides which are restricted by haploptypes other than A1 should increase the opportunity for paptide based immunotherapy. Conclusions This study shows that MAGE-1 mRNA is highly expressed in HCC tumor tissue in Chinese patients. Previously unreported point mutations in the MAGE-1 gene are described and may also provide additional opportunities for immunotherapy.