Application of Prenatal Whole Exome Sequencing for Structural Congenital Anomalies-Experience from a Local Prenatal Diagnostic Laboratory.

Fetal structural congenital abnormalities (SCAs) complicate 2-3% of all pregnancies. Whole-exome sequencing (WES) has been increasingly adopted prenatally when karyotyping and chromosomal microarray do not yield a diagnosis. This is a retrospective cohort study of 104 fetuses with SCAs identified on antenatal ultrasound in Hong Kong, where whole exome sequencing is performed. Molecular diagnosis was obtained in 25 of the 104 fetuses (24%). The highest diagnostic rate was found in fetuses with multiple SCAs (29.2%), particularly those with involvement of the cardiac and musculoskeletal systems. Variants of uncertain significance were detected in 8 out of the 104 fetuses (7.7%). Our study shows the utility of WES in the prenatal setting, and the extended use of the technology would be recommended in addition to conventional genetic workup.

PinX1t, a Novel PinX1 Transcript Variant, Positively Regulates Cardiogenesis of Embryonic Stem Cells.

Background Pin2/TRF1-interacting protein, PinX1, was previously identified as a tumor suppressor. Here, we discovered a novel transcript variant of mPinX1 (mouse PinX1), mPinX1t (mouse PinX1t), in embryonic stem cells (ESCs). The aims of this investigation were (1) to detect the presence of mPinX1 and mPinX1t in ESCs and their differentiation derivatives; (2) to investigate the role of mPinX1 and mPinX1t on regulating the characteristics of undifferentiated ESCs and the cardiac differentiation of ESCs; (3) to elucidate the molecular mechanisms of how mPinX1 and mPinX1t regulate the cardiac differentiation of ESCs. Methods and Results By 5' rapid amplification of cDNA ends, 3' rapid amplification of cDNA ends, and polysome fractionation followed by reverse transcription-polymerase chain reaction, mPinX1t transcript was confirmed to be an intact mRNA that is actively translated. Western blot confirmed the existence of mPinX1t protein. Overexpression or knockdown of mPinX1 (both decreased mPinX1t expression) both decreased while overexpression of mPinX1t increased the cardiac differentiation of ESCs. Although both mPinX1 and mPinX1t proteins were found to bind to cardiac transcription factor mRNAs, only mPinX1t protein but not mPinX1 protein was found to bind to nucleoporin 133 protein, a nuclear pore complex component. In addition, mPinX1t-containing cells were found to have a higher cytosol-to-nucleus ratio of cardiac transcription factor mRNAs when compared with that in the control cells. Our data suggested that mPinX1t may positively regulate cardiac differentiation by enhancing export of cardiac transcription factor mRNAs through interacting with nucleoporin 133. Conclusions We discovered a novel transcript variant of mPinX1, the mPinX1t, which positively regulates the cardiac differentiation of ESCs.

Fracture risk and bone mineral density reduction associated with proton pump inhibitors.

Many patients receive prolonged proton pump inhibitor (PPI) therapy for upper gastrointestinal disorders, but the long-term safety of PPIs, particularly increased risk of hip and nonhip fractures, has been questioned. To summarize the current literature on the risk of bone mineral density (BMD) reduction and fracture associated with PPI therapy, we conducted a literature search to identify all pertinent studies from 1980-February 2011. A total of 14 observational studies were included in this review. Most studies evaluated the risk of fracture associated with prolonged PPI exposure. Eight studies found an increased fracture risk at the hip, and five studies found an increased fracture risk at the spine associated with PPIs. Three studies showed reduction in fracture risk associated with PPIs after discontinuation for 1 month-1 year. Three studies evaluated the risk of BMD reduction associated with PPIs but did not find consistent changes in baseline or subsequent BMD. The current data suggest a modest increase in the risk of hip fracture and vertebral fracture associated with PPIs, although some studies showed conflicting results. Further studies will be needed to determine whether the increased risk of fracture is due to PPI exposure or residual confounding.

Effects of hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blockers on the proliferation and cell cycle progression of embryonic stem cells.

Embryonic stem cells (ESCs) can uniquely proliferate indefinitely and differentiate into all cell lineages. ESCs may therefore provide an unlimited supply of cells for cell-based therapies. Previous study reported the presence of hyperpolarization-activated inward currents in undifferentiated mouse (m) ESCs, but the functional role of this hyperpolarization-activated current in mESCs is unknown. In this study, the role of this current in maintaining the proliferative capacity and the cell cycle progression of ESCs was investigated. In D3 mESCs, this hyperpolarization-activated inward current can be blocked by HCN channel blocker ZD7288. Application of the HCN channel blockers, cesium (1-10 mM) or ZD7288 (0.1-30 µM), attenuated cell proliferation in a concentration-dependent manner. Both HCN blockers were found to be non-cytotoxic to mESCs as determined by cell viability test. Interestingly, ZD7288 at 10 and 30 µM was found to decrease the proportion of cells in G(0)/G(1) phase and increase the proportion of cells in S phase. This suggests that this hyperpolarization-activated current can affect the cell cycle progression in mESCs. In summary, the present investigation suggests that ESC proliferation and cell cycle progression can be regulated by this hyperpolarization-activated current.

Role of voltage-gated potassium channels in the fate determination of embryonic stem cells.

Embryonic stem cells (ESCs) possess two unique characteristics: self-renewal and pluripotency. In this study, roles of voltage-gated potassium channels (K(v)) in maintaining mouse (m) ESC characteristics were investigated. Tetraethylammonium (TEA(+)), a K(v) blocker, attenuated cell proliferation in a concentration-dependent manner. Possible reasons for this attenuation, including cytotoxicity, cell cycle arrest and differentiation, were examined. Blocking K(v) did not change the viability of mESCs. Interestingly, K(v) inhibition increased the proportion of cells in G(0)/G(1) phase and decreased that in S phase. This change in cell cycle distribution can be attributed to cell cycle arrest or differentiation. Loss of pluripotency as determined at both molecular and functional levels was detected in mESCs with K(v) blockade, indicating that K(v) inhibition in undifferentiated mESCs directs cells to differentiate instead of to self-renew and progress through the cell cycle. Membrane potential measurement revealed that K(v) blockade led to depolarization, consistent with the role of K(v) as the key determinant of membrane potential. The present results suggest that membrane potential changes may act as a "switch" for ESCs to decide whether to proliferate or to differentiate: hyperpolarization at G(1) phase would favor ESCs to enter S phase while depolarization would favor ESCs to differentiate. Consistent with this notion, S-phase-synchronized mESCs were found to be more hyperpolarized than G(0)/G(1)-phase-synchronized mESCs. Moreover, when mESCs differentiated, the differentiation derivatives depolarized at the initial stage of differentiation. This investigation is the first study to provide evidence that K(v) and membrane potential affect the fate determination of ESCs.