Clinical spectrum of SIX3-associated mutations in holoprosencephaly: correlation between genotype, phenotype and function.

BACKGROUND: Holoprosencephaly (HPE) is the most common structural malformation of the human forebrain. There are several important HPE mutational target genes, including the transcription factor SIX3, which encodes an early regulator of Shh, Wnt, Bmp and Nodal signalling expressed in the developing forebrain and eyes of all vertebrates. OBJECTIVE: To characterise genetic and clinical findings in patients with SIX3 mutations. METHODS: Patients with HPE and their family members were tested for mutations in HPE-associated genes and the genetic and clinical findings, including those for additional cases found in the literature, were analysed. The results were correlated with a mutation-specific functional assay in zebrafish. RESULTS: In a cohort of patients (n = 800) with HPE, SIX3 mutations were found in 4.7% of probands and additional cases were found through testing of relatives. In total, 138 cases of HPE were identified, 59 of whom had not previously been clinically presented. Mutations in SIX3 result in more severe HPE than in other cases of non-chromosomal, non-syndromic HPE. An over-representation of severe HPE was found in patients whose mutations confer greater loss of function, as measured by the functional zebrafish assay. The gender ratio in this combined set of patients was 1.5:1 (F:M) and maternal inheritance was almost twice as common as paternal. About 14% of SIX3 mutations in probands occur de novo. There is a wide intrafamilial clinical range of features and classical penetrance is estimated to be at least 62%. CONCLUSIONS: Our data suggest that SIX3 mutations result in relatively severe HPE and that there is a genotype-phenotype correlation, as shown by functional studies using animal models.

Nitric oxide causes apoptosis in pulmonary vascular smooth muscle cells.

Nitric oxide (NO), a product of certain cytokine-activated cells, affects rates of apoptosis, a mechanism of programmed cell death. We asked whether NO affected rates of apoptosis in pulmonary vascular cells. Using rat pulmonary artery smooth muscle cells, we studied direct effects of the NO donor SG-nitroso-acetyl-D,L-penicillamine (SNAP) and the effects of NO endogenously synthesized in response to bacterial lipopolysaccharide (LPS) and inflammatory cytokines interleukin-1beta, interferon-gamma, and tumor necrosis factor-alpha (a combination called cytomix for convenience). We determined apoptosis on the basis of light microscopy and the bromodeoxyuridine terminal deoxynucleotidyl transferase reaction (BrdUTdT). Both SNAP- and cytomix-induced synthesis of NO resulted in histologic evidence of apoptosis based upon fluorescence microscopy using propidium iodide. SNAP (10(-5) M) increased BrdUTdT-positive cells from 17.5 to 78.4% compared with basal medium alone, with the maximal response occurring at 15 h or exposure. Exposing cells to LPS and cytokines induced NO production (from 0.1 +/- 0.1 to 24.6 +/- 0.5 microM, P < 0.05) caused cytological changes consistent with apoptosis and led to an increase of increased BrdUTdT-positive cells from 11 to 41% at 12 h compared with basal medium alone. The competitive NO synthase inhibitor NG-monomethyl-L-arginine inhibited both NO synthesis and NO apoptosis, returning the proportion of BrdUTdT-positive cells (6%) to levels below control. L-Arginine (0.5 mM) restored percentages to those increase in response to endogenously synthesized NO, and NO is a potential mechanism of acute lung injury in response to inflammatory cytokines.

Liposome-cell interactions in vitro: effect of liposome surface charge on the binding and endocytosis of conventional and sterically stabilized liposomes.

The cellular uptake of liposomes is generally believed to be mediated by adsorption of liposomes onto the cell surface and subsequent endocytosis. This report examines the effect of liposome surface charge on liposomal binding and endocytosis in two different cell lines: a human ovarian carcinoma cell line (HeLa) and a murine derived mononuclear macrophage cell line (J774). The large unilamellar liposomes were composed of 1, 2-dioleolyl-sn-glycero-3-phosphatidylcholine with and without the addition of either a positively charged lipid, 1, 2-dioleoyl-3-dimethylammonium propanediol (DODAP), or a negatively charged lipid, 1,2-dioleolyl-sn-glycero-3-phosphatidylserine. In some experiments 5 mol % of the anionic PEG2000-PE or a neutral PEG lipid of the same molecular weight was added. HeLa cells were found to endocytose positively charged liposomes to a greater extent than either neutral or negatively charged liposomes. This preference was not lipid-specific since inclusion of a cationic cyanine dye, DiIC18(3), to impart positive charge in place of DODAP resulted in a similar extent of endocytosis. In contrast the extent of liposome interaction with J774 cells was greater for both cationic and anionic liposomes than for neutral liposomes. The greater uptake of positively charged liposomes by HeLa cells was also observed with sterically stabilized liposomes (PEG liposomes). Although the overall amount of endocytosis for all the PEG liposomes examined was attenuated relative to conventional liposomes, the extent of endocytosis was greatest for positively charged PEG liposomes, whereas negatively charged PEG2000-PE liposomes were hardly endocytosed by the HeLa cells. Incorporation of a neutral PEG lipid into liposomes permits the independent variation of liposome steric and electrostatic effects in a manner that may allow interactions with cells of the reticuloendothelial system to be minimized, yet permit strong interactions between liposomes and proliferating cells.

Velo-cardio-facial syndrome. Intrafamilial variability of the phenotype.

We describe a mother and son with velo-cardio-facial syndrome (VCFS) in whom cytogenetic and DNA molecular studies demonstrate an interstitial deletion of the long arm of chromosome 22. Although these two individuals manifest the typical facial and cognitive features of VCFS, they are discordant for the cardiovascular and palatal anomalies, which are seminal manifestations of the disorder. Previously, this degree of phenotypic variability had not been well appreciated within a single family segregating the VCFS deletion. A review of other familial cases of VCFS suggests that the family described in this article is not atypical. Because a microdeletion would be expected to be inherited without alteration within individual families, the phenotypic variability observed in these families appears to be an intrinsic quality of the syndrome and not wholly due to genetic heterogeneity.