Clinical and genetic study of 46 Italian patients with primary lymphedema.

Primary lymphedema is characterized by altered morphological development of lymphatic vessels causing fluid accumulation in interstitial spaces. In familial forms, it is primarily transmitted as a dominant Mendelian trait with heterozygous mutations in genes involved in lymphangiogenesis. We used PCR and direct sequencing to analyze the region of the fms-related tyrosine kinase 4 (FLT4) gene encoding the "tyrosine-kinase domain" and the single exon of the forkhead box C2 (FOXC2) gene in 46 Italian probands with primary lymphedema, 42 of whom had familial forms. We identified 12 mutations in 12 patients (12/46, 26%), six in the FLT4 gene and six in the FOXC2 gene. Most of the mutations (9/12, 75%) were new, and none were identified in 100 healthy subjects or listed in the NCBI dbSNP. A clear relation emerged between genotype and phenotype because 4/5 (80%) probands with onset at birth showed FLT4 mutations and 4/5 (80%) probands without distichiasis and with FOXC2 mutations had an amino-acid substitution outside the forkhead domain. Besides the allelic heterogeneity shown by unique mutations in each proband, the absence of mutations in almost 75% of familial cases of primary lymphedema also suggests genetic heterogeneity.

SVEP1 is important for morphogenesis of lymphatic system: Possible implications in lymphedema.

SVEP1, also known as Polydom, is a large extracellular mosaic protein with functions in protein interactions and adhesion. Since Svep1 knockout animals show severe edema and lymphatic system malformations, the aim of this study is to evaluate the presence of SVEP1 variants in patients with lymphedema. We analyzed DNA from 246 lymphedema patients for variants in known lymphedema genes, 235 of whom tested negative and underwent a second testing for new candidate genes, including SVEP1, as reported here. We found three samples with rare heterozygous missense single-nucleotide variants in the SVEP1 gene. In one family, healthy members were found to carry the same variants and reported some subclinical edema. Based on our findings and a review of the literature, we propose SVEP1 as a candidate gene that should be sequenced in patients with lymphatic malformations, with or without lymphedema, in order to investigate and add evidence on its possible involvement in the development of lymphedema.

Review of the function of SEMA3A in lymphatic vessel maturation and its potential as a candidate gene for lymphedema: Analysis of three families with rare causative variants.

SEMA3A is a semaphorin involved in cell signaling with PlexinA1 and Neuropilin-1 (NRP1) receptors and it is responsible for recruiting dendritic cells into lymphatics. Mutations in the SEMA3A gene result in abnormalities in lymphatic vessel development and maturation. We investigated the association of SEMA3A variants detected in lymphedema patients with lymphatic maturation and lymphatic system malfunction. First, we used NGS technology to sequence the SEMA3A gene in 235 lymphedema patients who carry wild type alleles for known lymphedema genes. We detected three different missense variants in three families. Bioinformatic results showed that some protein interactions could be altered by these variants. Other unaffected family members of the probands also reported different episodes of subclinical edema. We then evaluated the importance of the SEMA3A gene in the formation and maturation of lymphatic vessels. Our results determined that SEMA3A variants segregate in families with lymphatic system malformations and recommend the inclusion of SEMA3A in the gene panel for testing of patients with lymphedema.

CYP26B1 and its implications in lymphangiogenesis: Literature review and study of rare variants in two families.

CYP26B1 is a member of the cytochrome P450 family and is responsible for the break-down of retinoic acid for which appropriate levels are important for normal development of the cardiovascular and lymphatic systems. In a cohort of 235 patients with lymphatic malformations, we performed genetic testing for the CYP26B1 gene. These probands had previously tested negative for known lymphedema genes. We identified two heterozygous missense CY-P26B1 variants in two patients. Our bioinformatic study suggested that alterations caused by these variants have no major effect on the overall stability of CYP26B1 protein structure. Balanced levels of retinoic acid maintained by CYP26B1 are crucial for the lymphatic system. We identified that CYP26B1 could be involved in predisposition for lymphedema. We propose that CYP26B1 be further explored as a new candidate gene for genetic testing of lymphedema patients.

Effects of bilirubin on potassium (86Rb+) influx and ionic content in Ehrlich ascites cells.

Potassium influx, intracellular potassium and sodium content and cellular volume were determined in vitro in Ehrlich ascites cells in the presence of up to 0.8 mM bilirubin in the incubation medium. Bilirubin uptake into cells as a function of bilirubin concentration in the incubation medium increased linearly with a molar bilirubin/albumin ratio of 20 : 1. Potassium influx and intracellular content decreased while cellular volume increased after 180 min of incubation of cells in bilirubin at a molar bilirubin/albumin ratio of 20 : 1. At a bilirubin/albumin ratio 2 : 1, potassium influx decreased, cellular volume remained unchanged, and bilirubin uptake into cells became saturated at bilirubin concentrations greater than 0.3 mM. It is suggested that bilirubin-induced alterations in potassium gradients across cell membranes may play a role in toxic effects of bilirubin on cells.

The homogeneous effect of calcium ionophore A23187 on potassium loss in human foetal red cell populations.

A "pulse like" increase of cytoplasmic calcium concentration, which is proportional to ionophore concentration, is induced in red cells by exposure to A23187. Different Ca2+ levels are attained depending on cellular calcium buffering power and/or primary active calcium transport activation. We examined the effect of A23187 concentration of potassium loss in neonatal (nRC) as well as in adult red cells (aRC). The increase in ionophore concentration produced an "all- or -none" recruitment in adult cells and a "gradual" one in neonatal red cells. The "gradual" response observed in nRC would suggest that the "all or none" character of the response is not present in red cells during the foetal stages of haematopoiesis.

Human red cells from prenatal hemopoiesis. Sodium/lithium exchange symmetry.

Human red blood cells from prenatal stages of hemopoiesis (umbilical cord blood) and with increased lithium content were submitted to media with varying sodium concentrations in order to characterize extracellular sodium dependent-lithium efflux. On the other hand cells with different sodium contents were submitted to lithium media in order to characterize cellular sodium dependent-lithium influx through the above system. Experimental data were fitted by simple Michaelis-Menten kinetics. The Km value for lithium efflux (extracellular sodium-dependent) was significantly lower than the Km value for lithium influx (cellular sodium -dependent). The Vmax value for lithium influx was significantly greater than that for lithium efflux. The Vmax/Km ratio values were independent from the way in which the fluxes were measured. It is concluded that the simple carrier cannot be excluded as a model of the Na+-Li+ exchange system in these cells.

Human red cells from pre (hepato splenic-late fetal) and postnatal (bone marrow-adult's) stages of haemopoiesis: Na+/Li+ exchange kinetic.

The kinetic parameters of Na(+)-Li+ exchange were studied in both neonatal and adult's red blood cells in order to evaluate if the asymmetry for Li+ fluxes (Na(+)-contralateral-dependent) is expressed in both types of cells. Maximum velocities (Vmax) and Km (half-activation constant) were measured for Li+ fluxes in both types of cells. In human neonatal red blood cells (nRBC), extracellular Na+-dependent Li+ efflux was shown to be a saturable function of external sodium concentration with high affinity (apparent Km: 2.1 mM) and low capacity (maximal velocity Vmax = 0.3 mmoles Li+ 1('''10 cells h(-1)). The Vmax and apparent Km for cellular Na(+)-dependent Li+ influx were higher (Km = 12.9 mM; Vmax = 1.07 mmoles Li+ 1(-1) cells h(-1)). The results provide evidence for intrinsic functional asymmetry in this transport system, as the transporter is more prevalent and stable in the inward-facing conformation. These kinetic observations may be explained in terms of the simple carrier transport model. Similar findings were found for this system in human adult's red cells. These results point to the asymmetry pattern (related to Na+ activation of Li+ flux) as developed in red cells from late prenatal stages of hemopoiesis.

Calcium ionophore (A23187) differential effect on red cells from pre and postnatal haemopoiesis.

Differences in ionic transport mechanisms through human red cell membranes from pre and postnatal haemopoietic stages of development have been related. The analysis of calcium-sensitive conductive potassium pathways [K+ (Ca2+)] was realized from intracellular calcium level increase after treatment of cells with ionophores. The effect of calcium ionophore (A23187) on potassium transport in human neonatal red cells (nRC) was studied and compared with red cells from adults (aRC). Analysis focused on intra/extracellular potassium redistribution and potassium conductance [K+ (Ca2+)] values in incubated red cells suspensions. A difference was observed between neonatal and adult's red cells only for the first of the parameters referred to. Conductance values of the same order of magnitude for these potassium transport pathways between both cell types suggest that the results presented could rest on different [K+ (Ca2+)] cellular density.

Human red blood cells from prenatal hemopoiesis. Lithium flux (sodium dependent) asymmetry.

In red cells from umbilical cord blood it has been referred the existence of lithium fluxes (contralateral sodium dependent) asymmetry. On account of the relevancy of this transport system in some pathologies it is pertinent the study of its kinetics to relate normal with pathological states in which it is affected. Lithium fluxes--contralateral sodium dependent were determined in N-ethylmaleimide treated neonatal red blood cells. Experimental data were fitted by simple Michaelis-Menten kinetics, finding Km and Vmax variables. It was shown the persistency of asymmetry. The independence of sulfhydryl groups (or the occultation of the groups involved to this inhibitor) could explain asymmetry persistence.

Foreign anion substitution for chloride in neonatal human red cells. Effect on cellular volume.

The effect of replacement of chloride by thiocyanate has been investigated in neonatal human red cells. On incubating these cells in isotonic SCN- medium, a rapid cellular swelling was observed, which was not evident in Cl- media. Incubation in hypertonic SCN- medium showed a rapid osmotic shrinkage followed by reswelling back to the initial volume. This regulatory volume increase was completed in a shorter time than in Cl- medium. Whole water accumulated by the cells in both experimental conditions can be accounted for by net Na+ uptake (twofold augmentation with respect to initial value). Amiloride inhibits specifically both cellular swelling and the Na+ content increase in these experimental conditions. Similar experiments in neonatal red cells incubated in NO3- media, as well as in adult red cells placed in NO3- or SCN- media, did not exhibit the same response. The findings suggest that the response of neonatal red cells in a SCN- containing media was mediated by activation of a Na+/H+ antiport mechanism.

Lead ions but not other metallic ions increase resistance to hypotonic lysis in prenatal hemopoiesis red blood cells.

Metals known to have toxic effects on exposed individuals (Aluminum (Al), Cadmium (Cd), Zinc (Zn) and lead (Pb)) were selected. Umbilical cord erythrocytes from normal newborns were incubated in isotonic media alone or with addition of Pb (20 microM), Cd, Zn or Al (concentration range: 20-250 microM). Red cells were then placed in media of diminishing tonicity, to measure cellular lysis and volume; the regression curves of percent lysis as a function of osmolarity were determined for each data set and the break points calculated. Resistance to lysis increased significantly in Pb treated cells whereas cells treated with the other metals did not differ from controls, even at concentrations ten times higher than that of Pb. Lead produced a reduction in cellular volume corrected by addition of quinidine (an inhibitor of potassium channels activation) to the cell suspension; on the other hand, quinidine did not modify the effect of lead on lysis sensitivity. These results suggest that the effect of lead on cell resistance to lysis might be mediated by changes in membrane structure. The other metals examined did not affect the variables studied.

Furosemide-sensitive sodium and potassium fluxes in human neonatal erythrocytes.

During the course of incubation, in vitro, in a saline medium, we found an increase of cell volume and Na+ content in human neonatal erythrocytes (NNE), from the umbilical cord. The increased cell volume was dependent on the major anion in the medium in that replacement of Cl- by NO-3 abolished the cell volume increase. In erythrocytes from adults neither the cell volume nor the sodium content were altered under similar incubation conditions. Furosemide-sensitive Na+ and K+ fluxes were at variance from those reported from adult erythrocytes. The differences here presented between both cell types would be another instance of changes observed to occur in erythrocytes during the postnatal period.

Neonatal red blood cells as a model for the study of ouabain-furosemide interaction on active K+ transport. A theoretical approach.

In a former paper by Serrani et al. (1990), an overlapping action of ouabain and furosemide on potassium influx was observed. In the present paper we demonstrated that Na, K-ATPase from neonatal red cells ghosts is a target of both drugs. Furthermore the combined action of ouabain and furosemide on potassium (86Rb) influx (in its greatest fraction mediated by primary active transport) was studied by generalized equations for the analysis of inhibitors. The isobologram and the combination index performed from the data obtained point to the existence of synergism or antagonism between both inhibitors according to the fraction affected. Clinical implications of these findings could be postulated.

Unconjugated bilirubin effect on 3H-ouabain binding to human fetal red cells.

Human fetal red cells show heterogeneity of 3H-ouabain binding sites. These cells were chosen as a model to look into unconjugated bilirubin effects on the primary active Na(+)-K+ transport mechanism. Evidences are presented suggesting that unconjugated bilirubin affects 3H-ouabain binding but not through a direct effect. This is supported by the fact that the "low affinity" subgroup sites of the last mentioned ligand persists after unconjugated bilirubin treatment of cells, whereas the "high-affinity" subgroup disappears.