Detecting 22q11.2 Deletion Syndrome in Newborns with Low T Cell Receptor Excision Circles from Severe Combined Immunodeficiency Screening.

OBJECTIVE: Based on experiences and results from newborn screening for severe combined immunodeficiency (SCID), we evaluated the occurrence of chromosome 22q11.2 deletion syndrome (22q11.2DS) in newborns with different T cell receptor excision circles (TREC) results and established a second tier genetic test for 22q11.2DS. STUDY DESIGN: Recalled dried blood spots from 486 newborns with TREC results <90 copies/uL were tested from the SCID newborn screening. Quantitative real-time polymerase chain reaction assay was used to detect the copy number of TBX1 and HIRA genes by simple DNA extraction method. Multiplex ligation dependent probe amplification was used for further confirmation. RESULTS: Four hundred sixty-eight cases were considered negative because their haploid copy number of TBX1 and HIRA genes was >0.75. Eighteen cases with TBX1 and/or HIRA gene copy number <0.75 were suspected as positive, and 13 cases were further confirmed with 22q11.2DS. Detection rates of 22q11.2DS were 10.7% (6/56) in TREC <30 copies, 6.8% (9/132) in <50 TREC copies, 4.6% (12/260) in <70 TREC copies, and 2.7% (13/486) in <90 TREC copies. CONCLUSIONS: 22q11.2DS detection can be incorporated into the second-tier assay in subjects with low TREC copies in SCID screening. The dried blood spot methods were feasible for 22q11.2DS newborn screening.

Cardiac mast cells cause atrial fibrillation through PDGF-A-mediated fibrosis in pressure-overloaded mouse hearts.

Atrial fibrillation (AF) is a common arrhythmia that increases the risk of stroke and heart failure. Here, we have shown that mast cells, key mediators of allergic and immune responses, are critically involved in AF pathogenesis in stressed mouse hearts. Pressure overload induced mast cell infiltration and fibrosis in the atrium and enhanced AF susceptibility following atrial burst stimulation. Both atrial fibrosis and AF inducibility were attenuated by stabilization of mast cells with cromolyn and by BM reconstitution from mast cell-deficient WBB6F1-KitW/W-v mice. When cocultured with cardiac myocytes or fibroblasts, BM-derived mouse mast cells increased platelet-derived growth factor A (PDGF-A) synthesis and promoted cell proliferation and collagen expression in cardiac fibroblasts. These changes were abolished by treatment with a neutralizing antibody specific for PDGF alpha-receptor (PDGFR-alpha). Consistent with these data, upregulation of atrial Pdgfa expression in pressure-overloaded hearts was suppressed by BM reconstitution from WBB6F1-KitW/W-v mice. Furthermore, injection of the neutralizing PDGFR-alpha-specific antibody attenuated atrial fibrosis and AF inducibility in pressure-overloaded hearts, whereas administration of homodimer of PDGF-A (PDGF-AA) promoted atrial fibrosis and enhanced AF susceptibility in normal hearts. Our results suggest a crucial role for mast cells in AF and highlight a potential application of controlling the mast cell/PDGF-A axis to achieve upstream prevention of AF in stressed hearts.

Multivalent ligand-receptor interactions elicit inverse agonist activity of AT(1) receptor blockers against stretch-induced AT(1) receptor activation.

Type 1 angiotensin II (AT(1)) receptor has a critical role in the development of load-induced cardiac hypertrophy. Recently, we showed that mechanical stretching of cells activates the AT(1) receptor without the involvement of angiotensin II (AngII) and that this AngII-independent activation is inhibited by the inverse agonistic activity of the AT(1) receptor blocker (ARB), candesartan. Although the inverse agonist activity of ARBs has been studied in terms of their action on constitutively active AT(1) receptors, the structure-function relationship of the inverse agonism they exert against stretch-induced AT(1) receptor activation has not been fully elucidated. Assays evaluating c-fos gene expression and phosphorylated extracellular signal-regulated protein kinases (ERKs) have shown that olmesartan has strong inverse agonist activities against the constitutively active AT(1) receptor and the stretch-induced activation of AT(1) receptor, respectively. Ternary drug-receptor interactions, which occur between the hydroxyl group of olmesartan and Tyr(113) and between the carboxyl group of olmesartan and Lys(199) and His(256), were essential for the potent inverse agonist action olmesartan exerts against stretch-induced ERK activation and the constitutive activity of the AT(1)-N111G mutant receptor. Furthermore, the inverse agonist activity olmesartan exerts against stretch-induced ERK activation requires an additional drug-receptor interaction involving the tetrazole group of olmesartan and Gln(257) of the AT(1) receptor. These results suggest that multivalent interactions between an inverse agonist and the AT(1) receptor are required to stabilize the receptor in an inactive conformation in response to the distinct processes that lead to an AngII-independent activation of the AT(1) receptor.

PDK1 coordinates survival pathways and beta-adrenergic response in the heart.

The 3-phosphoinositide-dependent kinase-1 (PDK1) plays an important role in the regulation of cellular responses in multiple organs by mediating the phosphoinositide 3-kinase (PI3-K) signaling pathway through activating AGC kinases. Here we defined the role of PDK1 in controlling cardiac homeostasis. Cardiac expression of PDK1 was significantly decreased in murine models of heart failure. Tamoxifen-inducible and heart-specific disruption of Pdk1 in adult mice caused severe and lethal heart failure, which was associated with apoptotic death of cardiomyocytes and beta(1)-adrenergic receptor (AR) down-regulation. Overexpression of Bcl-2 protein prevented cardiomyocyte apoptosis and improved cardiac function. In addition, PDK1-deficient hearts showed enhanced activity of PI3-Kgamma, leading to robust beta(1)-AR internalization by forming complex with beta-AR kinase 1 (betaARK1). Interference of betaARK1/PI3-Kgamma complex formation by transgenic overexpression of phosphoinositide kinase domain normalized beta(1)-AR trafficking and improved cardiac function. Taken together, these results suggest that PDK1 plays a critical role in cardiac homeostasis in vivo by serving as a dual effector for cell survival and beta-adrenergic response.