Characterization of multi-locus imprinting disturbances and underlying genetic defects in patients with chromosome 11p15.5 related imprinting disorders.

The identification of multilocus imprinting disturbances (MLID) appears fundamental to uncover molecular pathways underlying imprinting disorders (IDs) and to complete clinical diagnosis of patients. However, MLID genetic associated mechanisms remain largely unknown. To characterize MLID in Beckwith-Wiedemann (BWS) and Silver-Russell (SRS) syndromes, we profiled by MassARRAY the methylation of 12 imprinted differentially methylated regions (iDMRs) in 21 BWS and 7 SRS patients with chromosome 11p15.5 epimutations. MLID was identified in 50% of BWS and 29% of SRS patients as a maternal hypomethylation syndrome. By next-generation sequencing, we searched for putative MLID-causative mutations in genes involved in methylation establishment/maintenance and found two novel missense mutations possibly causative of MLID: one in NLRP2, affecting ADP binding and protein activity, and one in ZFP42, likely leading to loss of DNA binding specificity. Both variants were paternally inherited. In silico protein modelling allowed to define the functional effect of these mutations. We found that MLID is very frequent in BWS/SRS. In addition, since MLID-BWS patients in our cohort show a peculiar pattern of BWS-associated clinical signs, MLID test could be important for a comprehensive clinical assessment. Finally, we highlighted the possible involvement of ZFP42 variants in MLID development and confirmed NLRP2 as causative locus in BWS-MLID.

Fetal growth patterns in Beckwith-Wiedemann syndrome.

We provide data on fetal growth pattern on the molecular subtypes of Beckwith-Wiedemann syndrome (BWS): IC1 gain of methylation (IC1-GoM), IC2 loss of methylation (IC2-LoM), 11p15.5 paternal uniparental disomy (UPD), and CDKN1C mutation. In this observational study, gestational ages and neonatal growth parameters of 247 BWS patients were compared by calculating gestational age-corrected standard deviation scores (SDS) and proportionality indexes to search for differences among IC1-GoM (n = 21), UPD (n = 87), IC2-LoM (n = 147), and CDKN1C mutation (n = 11) patients. In IC1-GoM subgroup, weight and length are higher than in other subgroups. Body proportionality indexes display the following pattern: highest in IC1-GoM patients, lowest in IC2-LoM/CDKN1C patients, intermediate in UPD ones. Prematurity was significantly more prevalent in the CDKN1C (64%) and IC2-LoM subgroups (37%). Fetal growth patterns are different in the four molecular subtypes of BWS and remarkably consistent with altered gene expression primed by the respective molecular mechanisms. IC1-GoM cases show extreme macrosomia and severe disproportion between weight and length excess. In IC2-LoM/CDKN1C patients, macrosomia is less common and associated with more proportionate weight/length ratios with excess of preterm birth. UPD patients show growth patterns closer to those of IC2-LoM, but manifest a body mass disproportion rather similar to that seen in IC1-GoM cases.

Effect of cyclic stretch on endothelial cells from different vascular beds.

Endothelial cells (EC) mediate many of the organ responses to shock. Much of our knowledge of EC are obtained from cell culture studies. However, compared to the dynamic milieu in vivo, the stationary environment for large-vessel EC may be artificial and inappropriate. In this study, the morphology, growth rate, and production of prostacyclin (PGI2) by EC obtained from different vascular beds under stationary and dynamic conditions were examined. EC were harvested from the thoracic aorta (Ao), pulmonary artery (PA), and vena cava (VC) of the same calves and exposed to 0.5 sec 24% deformation alternating with 0.5 sec relaxation (i.e., 60 cycles/min). Our results show that in response to the cyclic regimen, VCEC were elongated perpendicular to the force vector and their actin filaments aligned in the same direction, while AoEC and PAEC did not exhibit any morphological changes. The growth rate of AoEC (but not PAEC or VCEC) was significantly enhanced when stimulated by cyclic stretch. In addition, AoEC demonstrated an increased PGI2 synthetic activity with cyclic stretch, while PAEC and VCEC were unaltered. We conclude that the maintenance of EC phenotype and function is dependent on the hemodynamic milieu in vivo and may be influenced by the vascular origin of the cultured EC.

Amelioration of renal ischemic injury by phosphocreatine.

Phosphocreatine (PCr) is a critical intracellular energy reservoir used in the regeneration of ATP. The aim of this study was to determine the efficacy of exogenously added PCr on preservation of renal function in an in vitro model. The renal artery and ureter of a rat were cannulated and the kidney was subjected to 45 min of normothermic in vivo ischemia. The kidneys were then perfused ex vivo with either a Krebs-bicarbonate solution (Krebs) or a Krebs solution containing 3 mM PCr or an osmotically balanced solution containing 3 mM PCr. Our results indicate that the perfusion of kidneys subjected to 45 min of warm ischemia with solutions containing PCr resulted in significant improvements in GFR, RPF, and V, FRNa and FRH2O compared to KREBS alone. This suggests that the important factor in preservation of kidney function after an initial ischemic insult may be the addition of PCr rather that the electrolyte solution used.

Stimulation of adenylate cyclase activity in cultured endothelial cells subjected to cyclic stretch.

While vascular endothelial cells are repeatedly stretched by the pulsatile nature of cardiac output, in vitro models traditionally used to study vascular biology involve static culture techniques. We have recently shown that pulsatile stretching of endothelial cells in culture will increase their rates of proliferation and regulate their secretion of macromolecules. The aim of this study was to determine whether membrane adenylate cyclase is involved in intracellular signalling during pulsatile stress. Bovine aortic endothelial cells were seeded on flexible-bottomed culture wells (3 x 10(5) cells/25 mm well) and allowed to attach for 48 hours. The culture wells were placed in a vacuum-operated stress providing instrument and subjected to 0.5 s of 24% strain, 0.5 s relaxation (60 cycles/min) for 0, 1, 3, 5, 7, 10 and 15 minutes (N = 24 wells/time point). Cells were homogenized and a crude membrane preparation (27,000 x g) was assayed for adenylate cyclase under basal and forskolin (100 microM) stimulated conditions. The results indicate that there is a time-dependent increase in both basal and stimulated adenylate cyclase with cyclic deformation and suggest that there may be a "stretch receptor" coupled to adenylate cyclase which can modulate endothelial cell function with hemodynamic changes.