Identification of novel rare copy number variants associated with sporadic tetralogy of Fallot and clinical implications.

Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart disease. Highly penetrant copy number variants (CNVs) and genes related to the etiology of TOF likely exist with differences among populations. We aimed to identify CNV contributions to sporadic TOF cases in Han Chinese. Genomic DNA was extracted from peripheral blood in 605 subjects (303 sporadic TOF and 302 unaffected Han Chinese [Control] from cardiac centers in China) and analyzed by genome-wide association study (GWAS). The GWAS results were compared with existing Database of Genetic Variants. These CNVs were further validated by qPCR. Bioinformatics analyses were performed with protein-protein interaction (PPI) network and KEGG pathway enrichment. Across all chromosomes 119 novel "TOF-specific CNVs" were identified with prevalence of CNVs of 21.5% in chromosomes 1-20 and 37.0% including Chr21/22. In chromosomes 1-20, CNVs on 11q25 (encompasses genes ACAD8, B3GAT1, GLB1L2, GLB1L3, IGSF9B, JAM3, LOC100128239, LOC283177, MIR4697, MIR4697HG, NCAPD3, OPCML, SPATA19, THYN1, and VPS26B) and 14q32.33 (encompasses genes THYN1, OPCML, and NCAPD3) encompass genes most likely to be associated with TOF. Specific CNVs found on the chromosome 21 (6.3%) and 22(11.9%) were also identified in details. PPI network analysis identified the genes covering the specific CNVs related to TOF and the signaling pathways. This study for first time identified novel TOF-specific CNVs in the Han Chinese with higher frequency than in Caucasians and with 11q25 and 14q32.33 not reported in TOF of Caucasians. These novel CNVs identify new candidate genes for TOF and provide new insights into genetic basis of TOF.

Egr1 deficiency disrupts dynamic equilibrium of chondrocyte extracellular matrix through PPARγ/RUNX2 signaling pathways.

BACKGROUND: This study is to investigate the effect of Egr1 on the mineralization and accumulation of chondrocyte extracellular matrix. METHODS: The femoral heads of patients of various heights were collected. Egr1 knockout mice were used. Their limb lengtha nd body weight were assessed. The bone characteristics were detected by micro-CT scan and histological staining. Immature murine articular chondrocytes (iMACs) were isolated. Gross morphology was observed by histological staining. Relevant mRNA and protein expression were detected by qRT-PCR and Western blot, respectively. the related proteins were observed by immunohistochemical staining and immunofluorescence assay. Chromatin immunoprecipitation and reporter gene assay were also used. TUNEL was used to detect apoptosis. RESULTS: It was found that shorter patients had reduced Egr1 expression levels in the hypertrophic cartilage zone of the femoral head. In addition, Egr1 knockout mice exhibited reduced body size. Micro-CT analysis showed that these mice also had reduced bone volume. Safranin-O staining showed that the extracellular matrix of these mice exhibited a relatively limited degree of mineralization, and TUNEL staining showed reduced cell apoptosis levels. After transfecting the iMACs with dominant-negative Egr1 adenoviruses to inhibit Egr1, the enzymes of Adamst4, Adamst5, Mmp3 and Mmp13 were significantly upregulated. ChIP and luciferase assays revealed that Egr1 might regulate the chondrocyte extracellular matrix by the PPARγ/RUNX2 signaling pathways. CONCLUSION: Egr1 has an important regulatory effect on the dynamic equilibrium of the chondrocyte extracellular matrix, which may be achieved through the PPARγ/RUNX2 signaling pathways.

Surgical treatment of tricuspid regurgitation after mitral valve surgery: a retrospective study in China.

BACKGROUND: Functional tricuspid regurgitation (TR) occurs in patients with rheumatic mitral valve disease even after mitral valve surgery. The aim of this study was to analyze surgical results of TR after previous successful mitral valve surgery. METHODS: From September 1996 to September 2008, 45 patients with TR after previous mitral valve replacement underwent second operation for TR. In those, 43 patients (95.6%) had right heart failure symptoms (edema of lower extremities, ascites, hepatic congestion, etc.) and 40 patients (88.9%) had atrial fibrillation. Twenty-six patients (57.8%) were in New York Heart Association (NYHA) functional class III, and 19 (42.2%) in class IV. Previous operations included: 41 for mechanical mitral valve replacement (91.1%), 4 for bioprosthetic mitral valve replacement (8.9%), and 7 for tricuspid annuloplasty (15.6%). RESULTS: The tricuspid valves were repaired with Kay's (7 cases, 15.6%) or De Vega technique (4 cases, 8.9%). Tricuspid valve replacement was performed in 34 cases (75.6%). One patient (2.2%) died. Postoperative low cardiac output (LCO) occurred in 5 patients and treated successfully. Postoperative echocardiography showed obvious reduction of right atrium and ventricle. The anterioposterior diameter of the right ventricle decreased to 25.5 ± 7.1 mm from 33.7 ± 6.2 mm preoperatively (P < 0. 05). CONCLUSION: TR after mitral valve replacement in rheumatic heart disease is a serious clinical problem. If it occurs or progresses late after mitral valve surgery, tricuspid valve annuloplasty or replacement may be performed with satisfactory results. Due to the serious consequence of untreated TR, aggressive treatment of existing TR during mitral valve surgery is recommended.

[Expression of PTEN, matrix metaloproteinases 2 and 9 in thymomas].

OBJECTIVE: To evaluate the expression of PTEN, MMP-2 and MMP-9 in thymomas and their correlation with clinical significance. METHODS: Immunohistochemical S-P assay was used to detect the expression of PTEN, MMP-2 and MMP-9 in 45 thymomas and 16 non-neoplastic thymuses. RESULTS: The positive rate of PTEN, MMP-2 and MMP-9 expression in 45 thymomas were 53.5%, 48.9% and 62.2%, respectively. There were significant differences between non-neoplastic thymus and thymomas (P < 0.05). The expression of PTEN, MMP-2 correlated with histological type (P < 0.05) and different clinical stage of the thymoma (P < 0.01). The expression of MMP-9 was not correlated with histological type and different clinical stages of thymoma (P > 0.05). CONCLUSION: PTEN, MMP-2 and MMP-9 may play an important role in the occurrence and development of thymoma. The expression of PTEN and MMP-2 correlates with the malignance and the aggression of thymoma.

The current strategy of repair of tetralogy of Fallot in children and adults.

OBJECTIVES: The strategies of repair of tetralogy of Fallot change with the age of patients. In children older than 4 years and adults, the optimal strategy may be to use different method of reconstruction of the right ventricular outflow tract from those followed in younger children, so as to avoid, or reduce, the pulmonary insufficiency that is increasingly known to compromise right ventricular function. METHODS: From April, 2001, through May, 2008, we undertook complete repair in 312 patients, 180 male and 132 female, with a mean age of 11.3 years +/-0.4 years, and a range from 4 to 48 years, with typical clinical and morphological features of tetralogy of Fallot, including 42 patients with the ventriculo-arterial connection of double outlet right ventricle. The operation was performed under moderate hypothermia using blood cardioplegia. The ventricular septal defect was closed with a Dacron patch. When it was considered necessary to resect the musculature within the right ventricular outflow tract, or perform pulmonary valvotomy, we sought to preserve the function of the pulmonary valve by protecting as far as possible the native leaflets, or creating a folded monocusp of autologous pericardium. RESULTS: The repair was achieved completely through right atrium in 192, through the right ventricular outflow tract in 83, and through the right atrium, the outflow tract, and the pulmonary trunk in 36 patients. A transjunctional patch was inserted in 169 patients, non-valved in all but 9. There were no differences regarding the periods of aortic cross-clamping or cardiopulmonary bypass. Of the patients, 5 died (1.6%), with no influence noted for the transjunctional patch. Of those having a non-valved patch inserted, three-tenths had pulmonary regurgitation of various degree, while those having a valved patch had minimal pulmonary insufficiency and good right ventricular function postoperatively, this being maintained after follow-up of 8 to 24-months. CONCLUSIONS: Based on our experience, we suggest that the current strategy of repair of tetralogy of Fallot in older children and adults should be based on minimizing the insertion of transjunctional patches, this being indicated only in those with very small ventriculo-pulmonary junctions. If such a patch is necessary, then steps should be taken to preserve the function of the pulmonary valve.

High throughput detection of microsatellite instability by denaturing high-performance liquid chromatography.

Microsatellite instability (MSI) is a hallmark of the DNA replication error phenotype, due to the inactivation of mismatch repair genes. MSI has been implicated in colon and many other gastrointestinal cancers. MSI usually can be analyzed by PCR amplification of microsatellite markers followed by electrophoresis and detected using autoradiography or fluorescence techniques. We report here a novel method for high-throughput detection of MSI using denaturing high performance liquid chromatography (DHPLC). Amplification of two mononucleotide markers (BAT25 and BAT26) by polymerase chain reaction (PCR) is followed by DHPLC analysis to display alteration in the length of repetitive sequences. These two markers were tested in 84 colorectal cancer samples confirmed to be 44 MSI-H and 40 MSI-L or MSS, previously defined by multiple microsatellite markers and/or by immunohistochemical analyses of MLH1 and MSH2 proteins. Among 44 MSI-H samples, sequence variations in BAT26 and BAT25 were detected in 44 (100%) and 43 (98%), respectively, while no sequence variation in the two markers was detected in 40 MSI-L or MSS samples. A total of 96 gastric cancers and their matched normal tissues were then analyzed for MSI-H using this method. Sequence variations in BAT26 and BAT25 were detected in nine (9.4%) samples. Seven of the nine cases were shown unstable at both BAT26 and BAT25; one each was unstable at BAT26 or BAT25. These results were confirmed by autoradiography analyses. Together, our results demonstrate high sensitivity and specificity of DHPLC in the analysis of sequence variations in BAT25 and BAT26 to determine MSI status. This simple, efficient, and high-throughput approach will facilitate analysis of MSI in large sample sets of any cancers.