Reported in vivo splice-site mutations in the factor IX gene: severity of splicing defects and a hypothesis for predicting deleterious splice donor mutations

Small consensus sequences have been defined for RNA splicing, but questions about splicing in humans remain unanswered. Analysis of germline mutations in the factor IX gene offers a highly advantageous system for studying the mutational process in humans. In a sample of 860 families with hemophilia B, 9% of independent mutations are likely to disrupt splicing as their primary mode of action. This includes 26 splicing mutations reported herein. When combined with the factor IX splice mutations reported by others, at least 104 independent mutations have been observed, 80 of which are single base substitutions within the splice donor and splice acceptor consensus sequences. After analysis of these mutations, the following inferences emerge: (1) the susceptibility of a splice donor sequence to deleterious mutation depends on the degree of similarity with the donor consensus sequence, suggesting a simple "5-6 hypothesis" for predicting deleterious vs. neutral mutations; (2) the great majority of mutations that disrupt the splice donor or splice acceptor sequences result in at least a 100-fold decrement in factor IX coagulant activity, indicating that the mutations at these sites generally function as an on/off switch; (3) mutations that create cryptic splice junctions or that shorten but do not interrupt the polypyrimidine tract in the splice acceptor sequence can reduce splicing by a variable amount; and (4) there are thousands of potential donor-acceptor consensus sequence combinations in the 38-kb factor IX gene region apparently not reduced by evolutionary selective pressure, presenting an apparent paradox; i.e., mutations in the donor and acceptor consensus sequences at intron/exon splice junctions can dramatically alter normal splicing, yet, appropriately spaced, good matches to the consensus sequences do not predispose to significant amounts of alternative splicing.     

The citalopram challenge test in patients with major depression and in healthy controls

Neuroendocrine challenge tests in depressed patients have revealed a blunted hormonal reaction to serotonergic stimuli. In the present study, citalopram was chosen as the serotonergic agent for neuroendocrine stimulation. Compared to earlier challenge agents, citalopram has the advantage of serotonergic selectivity, its application is well tolerated and the possibility of intravenous application reduces pharmacokinetic interference. Sixteen patients suffering from an acute episode of major depression and 16 healthy controls underwent the stimulation procedure with 20 mg of citalopram and placebo. Whereas significant differences in the secretion of prolactin and cortisol between citalopram and placebo challenge were observed in the control group, no differences were found in the group of depressed patients. Comparison of depressed patients and controls showed a significantly blunted prolactin secretion in patients. Differences in cortisol secretion following serotonergic stimulation with citalopram did not become significant. The stimulation procedure was well tolerated in all subjects, although a higher number of side effects was observed in the control group. The amount of side effects did not correlate with the hormone responses. These results are in line with the hypothesis of serotonergic hypofunction in depressed patients. In conclusion, the 20-mg citalopram challenge test is thought to be a promising tool for further investigation of serotonergic function in psychiatric illness.     

Immunohistochemical evidence for the occurrence of similar epithelial phenotypes during lung development and radiation-induced fibrogenesis

PURPOSE: Processes of fibrosis, wound healing and tissue regeneration have in common the fact that proliferation and differentiation of cells involved in the restoration of normal-tissue architecture resemble to a certain degree the embryonic development of the corresponding tissue. The present review focuses on the phenotypic changes of alveolar epithelial cells during fibrogenesis and describes similarities in the expression pattern of epithelial antigens during lung development. METHODS: For comparative studies, immunohistochemical investigations of different experimental fibrosis models were performed. RESULTS: For several epithelial proteins, such as the CD44 adhesion molecule, the enzymes carbanhydrase II and cathepsin D, as well as the lectin galectin-3, a transient epithelial immunoreactivity have been detected. What all four examples have in common is that a part of the foetal antigenic profile reappears under conditions of injury and during the development of pulmonary fibrosis. CONCLUSIONS: The re-expression of foetal antigens in fibrotic samples with a spatio-temporal pattern, as detected by immunocytochemical techniques, indicates that some mechanisms or factors exist, which similarly regulate the differentiation of the epithelium during ontogenesis and in the remodelling process during fibrogenesis.     

The Relationship Between Cerebral Blood Flow Velocities and the Amount of Blood Clots in Computed Tomography After Subarachnoid Haemorrhage

127 patients with aneurysmal subarachnoid haemorrhage (SAH) were analyzed for the relationship between the amount of blood clots as detected by initial computed tomography (CT) up to 48 hours after SAH and changes of blood flow velocities as measured using transcranial Doppler ultrasonography (TCD). All patients were operated on within 72 hours after SAH. Patients who presented with remarkable brain oedema or with pathological intracranial pressure (ICP) due to mass effects of a haematoma, and who were in a poor neurological condition classified according to Hunt-Hess as grade V were excluded from this study. Serial TCD examination of the middle cerebral arteries (MCA) and anterior cerebral arteries (ACA) started within 48 hours after SAH and were performed daily up to three weeks. A statistically significant correlation between blood load designated according to Fisher's grading as group CT I-CT IV and mean flow velocities (MFV) was found in groups CT I, II, and III. High values of MFV in MCA examinations were noted in patients with severe SAH (group CT III)--161 cm/s, and low values in patients without SAH (group CT I)--119 cm/s. Patients with haematocephalus and/or haematoma without a mass effect (group CT IV) had lower blood flow velocities than patients with severe SAH (group CT III) but values were higher than in patients without SAH (group CT I). The number of days for which MFV in the MCA was > 120 cm/s and was statistically (p < 0.05) correlated with the amount of blood clots as observed in the respective CT (in group CT I, II, and III). MFV values in the anterior cerebral artery (ACA) were lower than those obtained in the middle cerebral artery (MCA) in all groups. Statistically significant (p < 0.05) differences were noted between groups CT I and CT III (first and third week) and between groups CT I and CT IV (third week). If the SAH was extensive in the CT scan, pathological values of MFV > 90 cm/s were observed in ACA, and this was more pronounced in group CT III than in group CT IV. Blood flow velocities obtained via TCD were registered to compare side-to-side differences and particularly high differences were observed in patients with severe SAH. It is concluded that the amount of blood clots in the initial computed tomography after SAH is significantly correlated with cerebral blood flow velocity measurements by TCD.     

First-trimester combined screening for Down syndrome: prediction of low birth weight, small for gestational age and pre-term delivery in a cohort of non-selected women

OBJECTIVE: To establish the relationship between the first-trimester screening markers [pregnancy-associated plasma protein A (PAPP-A), free human chorionic gonadotrophin-beta (beta-hCG), nuchal translucency (NT)], the Down syndrome (DS) risk estimate, and the adverse outcomes such as low birth weight, small for gestational age (SGA) and pre-term delivery. METHODS: A retrospective cohort study including 1,734 non-selected singleton pregnancies consecutively enrolled into the programme of first-trimester combined screening for DS in a 12-month period at a single centre. Data from the Prenatal Patient Registry in ASTRAIA were combined with the Danish National Newborn Screening Registry and Danish Birth Registry. RESULTS: There was a significant relation between low PAPP-A MoM, low beta-hCG MoM, increased risk estimate for DS and low birth weight and SGA. Low PAPP-A MoM and increased NT showed a significant relation to pre-term and spontaneous pre-term delivery. Low PAPP-A MoM showed a significant relation to early pre-term delivery. CONCLUSION: First-trimester screening markers exhibited a significant relation to low birth weight, SGA and to some extent, to pre-term and early pre-term delivery. The screening performance of individual markers was poor.