多切面法联合彩色多普勒超声在胎儿先天性心脏病诊断中的应用

目的:探讨多切面法联合彩色多普勒超声在胎儿先天性心脏病(congenital heart diseases,CHD)诊断中的应用价值。方法:采用多切面法联合彩色多普勒超声对2015年5月~2016年7月300例胎儿进行CHD筛查,并与随访的产后超声或尸解结果作对照。结果:300例胎儿经产前超声联合多切面法检出CHD胎儿20例,检出率为6.7%,经产后超声或尸解确诊14例:三尖瓣下移畸形1例,室间隔完整型完全性大动脉转位1例,完全性房室间隔缺损1例,室间隔完整型肺动脉瓣闭锁1例,双流入型单心室1例,共同动脉干Ⅰ型2例,单纯室间隔缺损2例,法洛氏四联症2例,主动脉弓缩窄1例,肺动脉瓣轻度狭窄1例,二尖瓣闭锁并共同动脉干1例;误诊为单纯室间隔缺损1例,误诊为法洛氏四联症1例,病例流失4例。产前超声联合多切面法对有、无高危因素的检出率分别为3.79%、13.48%,比较有统计学意义(P0.05)。产前超声联合多切面法诊断CHD的灵敏度为100%、特异度为99.66%、阳性预测值为80.00%、阴性预测值为100%。结论:多切面法联合彩色多普勒超声在胎儿CHD诊断中具有较高的应用价值。     

The F box protein S phase kinase-associated protein 2 regulates adipose mass and adipocyte number in vivo

Objective: The etiology of some obesity may involve adipocyte hyperplasia. However, the role of adipocyte number in establishing adipose mass is unclear. Cyclin‐dependent kinase inhibitor p27 regulates activity of cyclin/cyclin‐dependent kinase complexes responsible for cell cycle progression. This protein is critical for establishing adult adipocyte number, and p27 knockout increases adult adipocyte number. The SCF (for Skp1‐Cullin‐F‐box protein) complex targets proteins such as p27 for ubiquitin‐proteosome degradation; the F box protein S phase kinase‐associated protein 2 (Skp2), a component of the SCF complex, specifically recognizes p27 for degradation. We used Skp2 knockout (Skp2^?/?) mice to test whether Skp2 loss decreased adipose mass and adipocyte number. Research Methods and Procedures: We measured body weight, adipose mass, adipocyte diameter and number, and glucose tolerance in wild‐type (WT), Skp2^?/?, and p27^?/?Skp2^?/? mice. Mouse embryo fibroblasts (MEFs) from WT and Skp2^?/? fetuses were differentiated to determine whether Skp2 directly affected adipogenesis. Results: Skp2^?/? mice had a 50% decrease in both subcutaneous and visceral fat pad mass and adipocyte number; these decreases exceeded those in body weight, kidney, or muscle. To test the hypothesis that Skp2 effects on adipocyte number involved p27 accumulation, we used p27^?/?Skp2^?/? double knockout mice. The Skp2^?/? decrements in adipocyte number and fat pad mass were totally reversed in p27^?/?Skp2^?/? mice. Adipogenesis was inhibited in MEFs from Skp2^?/? vs. WT mice, and this inhibition was absent in MEFs from p27^?/?Skp2^?/? mice. Discussion: Our results indicate that Skp2 regulates adipogenesis and ultimate adipocyte number in vivo; thus, Skp2 may contribute to obesity involving adipocyte hyperplasia.     

Generation of mice carrying a knockout‐first and conditional‐ready allele of transforming growth factor beta2 gene

Transforming growth factor beta2 (TGFβ2) is a multifunctional protein which is expressed in several embryonic and adult organs. TGFB2 mutations can cause Loeys Dietz syndrome, and its dysregulation is involved in cardiovascular, skeletal, ocular, and neuromuscular diseases, osteoarthritis, tissue fibrosis, and various forms of cancer. TGFβ2 is involved in cell growth, apoptosis, cell migration, cell differentiation, cell‐matrix remodeling, epithelial‐mesenchymal transition, and wound healing in a highly context‐dependent and tissue‐specific manner. Tgfb2^?/? mice die perinatally from congenital heart disease, precluding functional studies in adults. Here, we have generated mice harboring Tgfb2^βgeo (knockout‐first lacZ‐tagged insertion) gene‐trap allele and Tgfb2^flox conditional allele. Tgfb2^βgeo/βgeo or Tgfb2^βgeo/‐ mice died at perinatal stage from the same congenital heart defects as Tgfb2^?/? mice. β‐galactosidase staining successfully detected Tgfb2 expression in the heterozygous Tgfb2^βgeo fetal tissue sections. Tgfb2^flox mice were produced by crossing the Tgfb2^+/βgeo mice with the FLPeR mice. Tgfb2^flox/? mice were viable. Tgfb2 conditional knockout (Tgfb2^cko/?) fetuses were generated by crossing of Tgfb2^flox/? mice with Tgfb2^+/?; EIIaCre mice. Systemic Tgfb2^cko/? embryos developed cardiac defects which resembled the Tgfb2^βgeo/βgeo, Tgfb2^βgeo/?, and Tgfb2^?/? fetuses. In conclusion, Tgfb2^βgeo and Tgfb2^flox mice are novel mouse strains which will be useful for investigating the tissue specific expression and function of TGFβ2 in embryonic development, adult organs, and disease pathogenesis and cancer. genesis 52:817–826, 2014. © 2014 Wiley Periodicals, Inc.     

Circadian rhythm in QT interval is preserved in mice deficient of potassium channel interacting protein 2

Potassium Channel Interacting Protein 2 (KChIP2) is suggested to be responsible for the circadian rhythm in repolarization duration, ventricular arrhythmias, and sudden cardiac death. We investigated the hypothesis that there is no circadian rhythm in QT interval in the absence of KChIP2. Implanted telemetric devices recorded electrocardiogram continuously for 5 days in conscious wild-type mice (WT, n = 9) and KChIP2^?/? mice (n = 9) in light:dark periods and in complete darkness. QT intervals were determined from all RR intervals and corrected for heart rate (QT₁₀₀ = QT/(RR/100)^1/2). Moreover, QT intervals were determined from complexes within the RR range of mean-RR ± 1% in the individual mouse (QT_mean-RR). We find that RR intervals are 125 ± 5 ms in WT and 123 ± 4 ms in KChIP2^?/? (p = 0.81), and QT intervals are 52 ± 1 and 52 ± 1 ms, respectively(p = 0.89). No ventricular arrhythmias or sudden cardiac deaths were observed. We find similar diurnal (light:dark) and circadian (darkness) rhythms of RR intervals in WT and KChIP2^?/? mice. Circadian rhythms in QT₁₀₀ intervals are present in both groups, but at physiological small amplitudes: 1.6 ± 0.2 and 1.0 ± 0.3 ms in WT and KChIP2^?/?, respectively (p = 0.15). A diurnal rhythm in QT₁₀₀ intervals was only found in WT mice. QT_mean-RR intervals display clear diurnal and circadian rhythms in both WT and KChIP2^?/?. The amplitude of the circadian rhythm in QT_mean-RR is 4.0 ± 0.3 and 3.1 ± 0.5 ms in WT and KChIP2^?/?, respectively (p = 0.16). In conclusion, KChIP2 expression does not appear to underlie the circadian rhythm in repolarization duration.     

Pax8 plays a pivotal role in regulation of cardiomyocyte growth and senescence

Congenital heart disease (CHD) is a worldwide health problem, particularly in young populations. In spite of the advancement and progress in medical research and technology, the underlying causative factors and mechanisms of CHD still remain unclear. Bone morphogenetic protein receptor IA (ALK3) mediates the development of ventricular septal defect (VSD). We have recently found that paired box gene 8 (Pax8) may be the downstream molecule of ALK3. Paired box gene 8 plays an essential role in VSD, and apoptosis and proliferation imbalance leads to septal dysplasia. Recent studies have also disclosed that cellular senescence also participates in embryonic development. Whether programmed senescence exists in cardiac organogenesis has not ever been reported. We hypothesized that together with various biological processes, such as apoptosis, enhanced cellular senescence may occur actively in the development of Pax8 null mice murine hearts. In H9C2 myogenic cells, Pax8 overexpression can rescue caspase‐dependent apoptosis induced by ALK3 silencing. Senescent cells and senescence‐associated mediators in Pax8 knockout hearts increased compared with the wild‐type ones in an age‐dependent manner. These results suggest that Pax8 maybe the downstream molecule of ALK3, it mediates the murine heart development perhaps via cellular senescence, which may serve as a mechanism that compensates for the cell loss via apoptosis in heart development.     

Maternal serum-derived exosomal lactoferrin as a marker in detecting and predicting ventricular septal defect in fetuses

Among different types of congenital heart diseases, ventricular septal defect is the most frequently diagnosed type and is frequently missed in early prenatal screening programs. Herein, we explored the role of maternal serum-derived exosomes in detecting and predicting ventricular septal defect in fetuses in the early stage of pregnancy. A total of 104 pregnant women consisting of 52 ventricular septal defect cases and 52 healthy controls were recruited. TMT/iTRAQ proteomic analysis uncovered 15 maternal serum exosomal proteins, which showed differential expression between ventricular septal defect and control groups. Among these, four down-regulated proteins, lactoferrin, SBSN, DCD, and MBD3, were validated by Western blot. The protein lactoferrin was additionally verified by ELISA which was able to distinguish ventricular septal defects from controls with area under the ROC curve (AUC) 0.804 (p < 0.001). Our findings reveal that lactoferrin in maternal serum-derived exosomes may be a potential biomarker for non-invasive prenatal diagnosis of fetal ventricular septal defects.     

Replication of the 4p16 Susceptibility Locus in Congenital Heart Disease in Han Chinese Populations

Congenital heart disease (CHD) is the most common form of congenital human birth anomalies and a leading cause of perinatal and infant mortality. Some studies including our published genome-wide association study (GWAS) of CHD have indicated that genetic variants may contribute to the risk of CHD. Recently, Cordell et al. published a GWAS of multiple CHD phenotypes in European Caucasians and identified 3 susceptibility loci (rs870142, rs16835979 and rs6824295) for ostium secundum atrial septal defect (ASD) at chromosome 4p16. However, whether these loci at 4p16 confer the predisposition to CHD in Chinese population is unclear. In the current study, we first analyzed the associations between these 3 single nucleotide polymorphisms (SNPs) at 4p16 and CHD risk by using our existing genome-wide scan data and found all of the 3 SNPs showed significant associations with ASD in the same direction as that observed in Cordell’s study, but not with other subtypes- ventricular septal defect (VSD) and ASD combined VSD. As these 3 SNPs were in high linkage disequilibrium (LD) in Chinese population, we selected one SNP with the lowest P value in our GWAS scan (rs16835979) to perform a replication study with additional 1,709 CHD cases with multiple phenotypes and 1,962 controls. The significant association was also observed only within the ASD subgroup, which was heterogeneous from other disease groups. In combined GWAS and replication samples, the minor allele of rs16835979 remained significant association with the risk of ASD (OR = 1.22, 95% CI = 1.08–1.38, P = 0.001). Our findings suggest that susceptibility loci of ASD identified from Cordell’s European GWAS are generalizable to Chinese population, and such investigation may provide new insights into the roles of genetic variants in the etiology of different CHD phenotypes.     

A critical role for CARD9 in pneumocystis pneumonia host defence

Caspase recruitment domains‐containing protein 9 (CARD9) is an adaptor molecule critical for key signalling pathways initiated through C‐type lectin receptors (CLRs). Previous studies demonstrated that Pneumocystis organisms are recognised through a variety of CLRs. However, the role of the downstream CARD9 adaptor signalling protein in host defence against Pneumocystis infection remains to be elucidated. Herein, we analysed the role of CARD9 in host defence against Pneumocystis both in CD4‐depleted CARD9^?/? and immunocompetent hosts. Card9 gene‐disrupted (CARD9^?/?) mice were more susceptible to Pneumocystis, as evidenced by reduced fungal clearance in infected lungs compared to wild‐type (WT) infected mice. Our data suggests that this defect was due to impaired proinflammatory responses. Furthermore, CARD9^?/? macrophages were severely compromised in their ability to differentiate and express M1 and M2 macrophage polarisation markers, to enhanced mRNA expression for Dectin‐1 and Mincle, and most importantly, to kill Pneumocystis in vitro. Remarkably, compared to WT mice, and despite markedly increased organism burdens, CARD9^?/? animals did not exhibit worsened survival during pneumocystis pneumonia (PCP), perhaps related to decreased lung injury due to altered influx of inflammatory cells and decreased levels of proinflammatory cytokines in response to the organism. Finally, although innate phase cytokines were impaired in the CARD9^?/? animals during PCP, T‐helper cell cytokines were normal in immunocompetent CARD9^?/? animals infected with Pneumocystis. Taken together, our data demonstrate that CARD9 has a critical function in innate immune responses against Pneumocystis.     

Real-Time 3-Dimensional Echocardiographic Assessment of Ventricular Volume,Mass, and Function in Human Fetuses

Objectives

We sought to determine the feasibility and reproducibility of real-time 3-dimensional echocardiography (RT3DE) for evaluation of cardiac volume, mass, and function and to characterize maturational changes of these measurements in human fetuses.

Methods

Eighty pregnant women in the 2^nd and 3^rd trimesters (59 with normal fetuses and 21 with fetuses with congenital heart disease [CHD]) were enrolled. We acquired RT3DE images using a matrix-array transducer. RT3DE measurements of volume, mass, stroke volume (SV), combined cardiac output (CCO), and ejection fraction (EF) were obtained. Images were scored and analyzed by two blinded independent observers. Inter- and intraobserver variabilities and correlations between fetal cardiac indices and gestational age were determined.

Results

Fifty-two of 59 normal data sets (88%) and 9 of 21 CHD data sets (43%) were feasible for analysis. In normal fetuses, the right ventricle (RV) is larger than the left ventricle (LV) (P<0.05), but no difference exists between the LV and RV in mass, SV, CO, and CO/CCO. The EFs for the LV and RV were diminished; the RVSV/LVSV was reduced in CHD fetuses compared with normal fetuses (P<0.05). Fetal ventricular volumes, mass, SV, and CCO fit best into exponential curves with gestational age, but LVEF, RVEF, and RVSV/LVSV remain relatively constant.

Conclusions

RT3DE is feasible and reproducible for assessment of LV and RV volume, mass, and function, especially in normal fetuses. Gestational growth of these measures, except for EF, is exponential in normal and CHD fetuses. CHD fetuses exhibit diminished LV and RV EFs.     

Bmp2 and Bmp4 genetically interact to support multiple aspects of mouse development including functional heart development

Bone morphogenetic proteins (BMPs) have multiple roles during embryogenesis. Current data indicate that the dosage of BMPs is tightly regulated for normal development in mice. Since Bmp2 or Bmp4 homozygous mutant mice show early embryonic lethality, we generated compound heterozygous mice for Bmp2 and Bmp4 to explore the impact of lowered dosage of these BMP ligands. Genotyping pups bred between Bmp2 and Bmp4 heterozygous mice revealed that the ratio of adult compound heterozygous mice for Bmp2 and Bmp4 is much lower than expected. During embryogenesis, the compound heterozygous embryos showed several abnormalities, including defects in eye formation, body wall closure defects, and ventricular septal defects (VSD) in the heart. However, the ratio of the compound heterozygous embryos was the same as expected. Caesarean sections at E18.5 revealed that half of the compound heterozygotes died soon after birth, and the majority of the dead individuals exhibited VSD. Survivors were able to grow to adults, but their body weight was significantly lower than control littermates. They demonstrated progressive abnormalities in the heart, eventually showing a branched leaflet in atrioventricular valves. These results suggest that the dosage of both BMP2 and 4 is critical for functional heart formation during embryogenesis and after birth. genesis 47:374–384, 2009. © 2009 Wiley‐Liss, Inc.     

Lack of maternal folic acid supplementation is associated with heart defects in Down syndrome: a report from the National Down Syndrome Project

BACKGROUND: Maternal folic acid supplementation has been associated with a reduced risk for neural tube defects and may be associated with a reduced risk for congenital heart defects and other birth defects. Individuals with Down syndrome are at high risk for congenital heart defects and have been shown to have abnormal folate metabolism. METHODS: As part of the population‐based case‐control National Down Syndrome Project, 1011 mothers of infants with Down syndrome reported their use of supplements containing folic acid. These data were used to determine whether a lack of periconceptional maternal folic acid supplementation is associated with congenital heart defects in Down syndrome. We used logistic regression to test the relationship between maternal folic acid supplementation and the frequency of specific heart defects correcting for maternal race or ethnicity, proband sex, maternal use of alcohol and cigarettes, and maternal age at conception. RESULTS: Lack of maternal folic acid supplementation was more frequent among infants with Down syndrome and atrioventricular septal defects (odds ratio [OR], 1.69; 95% confidence interval [CI], 1.08–2.63; p = 0.011) or atrial septal defects (OR, 1.69; 95% CI, 1.11–2.58; p = 0.007) than among infants with Down syndrome and no heart defect. Preliminary evidence suggests that the patterns of association differ by race or ethnicity and sex of the proband. There was no statistically significant association with ventricular septal defects (OR, 1.26; 95% CI, 0.85–1.87; p = 0.124). CONCLUSIONS: Our results suggest that lack of maternal folic acid supplementation is associated with septal defects in infants with Down syndrome. Birth Defects Research (Part A), 2011. © 2011 Wiley‐Liss, Inc.