Mutation analysis in Norwegian families with hereditary hemorrhagic telangiectasia: founder mutations in ACVRL1.

Hereditary hemorrhagic telangiectasia (HHT, Osler-Weber-Rendu disease) is an autosomal dominant inherited disease defined by the presence of epistaxis and mucocutaneous telangiectasias and arteriovenous malformations (AVMs) in internal organs. In most families (~85%), HHT is caused by mutations in the ENG (HHT1) or the ACVRL1 (HHT2) genes. Here, we report the results of genetic testing of 113 Norwegian families with suspected or definite HHT. Variants in ENG and ACVRL1 were found in 105 families (42 ENG, 63 ACVRL1), including six novel variants of uncertain pathogenic significance. Mutation types were similar to previous reports with more missense variants in ACVRL1 and more nonsense, frameshift and splice-site mutations in ENG. Thirty-two variants were novel in this study. The preponderance of ACVRL1 mutations was due to founder mutations, specifically, c.830C>A (p.Thr277Lys), which was found in 24 families from the same geographical area of Norway. We discuss the importance of founder mutations and present a thorough evaluation of missense and splice-site variants.

Seven novel mutations and four exon deletions in a collection of Norwegian patients with SPG4 hereditary spastic paraplegia.

To establish the phenotypic variation and frequency of SPAST mutations or deletions in Norwegian patients with hereditary spastic paraplegia (HSP), we examined 59 unrelated patients with HSP and screened for DNA point mutations and microdeletions in SPG4. Forty-one had a familial history, 35 had a clear dominant inheritance, six had other affected sibs and 18 were sporadic. We found 12 mutations in SPG4, seven of them novel, and four different heterozygous exon deletions, two of them novel. Mutations were found in 16 families showing autosomal dominant (AD) inheritance, and in one sporadic case. In two non-SPG4 families the S44L polymorphism/modifier was found in both affected and unaffected individuals. This is the first study of Norwegian patients with HSP since the 1970s, and the first report on SPG4 in Norway. Our results show that SPG4 mutations and deletions are a significant cause of HSP in our population and warrant SPG4 screening in AD families and selected sporadic cases.

Carnitine uptake into human heart cells in culture.

The uptake of radiolabeled carnitine and butyrobetaine has been studied in human heart cells (CCL 27). The uptake of carinitine is 3-10-fold higher in heart cells than in fibroblasts (pmol - mug DNA-1). The uptake of carnitine increases with temperature coefficient KT of 1.6 in the interval 10-20 degrees C and with a negligible uptake at 4 and 10 degrees C. The uptake of carnitine follows Michaelis-Menten kinetics with a KM of 4.8 +/- 2.2 muM and V = 8.7 +/- 3.2 pmol - mug DNA-1 - H-1. Carnitine uptake is suppressed 90% by NaF (24MM). Butyrobetaine is taken up into heart cells to the same extent as carnitine with a KM of 5.7-17.3 muM and V = 8.7-9.3 pmol - mug DNA-1 - h-1. Butyrobetaine inhibits competitively the uptake of carnitine and carnitine inhibits the uptake of butyrobetaine to the same extent. No conversion of radiolabeled butyrobetaine to carnitine, or carnitine to methyl choline was observed intra- or extracellulary during incubation. These data are compatible with a selective transport mechanism for carnitine which is also responsible for the uptake of butyrobetaine.

Non-random X chromosome inactivation in an affected twin in a monozygotic twin pair discordant for Wiedemann-Beckwith syndrome.

Wiedemann-Beckwith syndrome (WBS) is a syndrome including exomphalos, macroglossia, and generalized overgrowth. The locus has been assigned to 11p15.5, and genomic imprinting may play a part in the expression of one or more genes involved. Most cases are sporadic. An excess of female monozygotic twins discordant for WBS have been reported, and it has been proposed that this excess could be related to the process of X chromosome inactivation. We have therefore studied X chromosome inactivation in 13-year-old monozygotic twin girls who were discordant for WBS. In addition, both twins had Tourette syndrome. The twins were monochorionic and therefore the result of a late twinning process. This has also been the case in previously reported discordant twin pairs with information on placentation. X chromosome inactivation was determined in DNA from peripheral blood cells by PCR analysis at the androgen receptor locus. The affected twin had a completely skewed X inactivation, where the paternal allele was on the active X chromosome in all cells. The unaffected twin had a moderately skewed X inactivation in the same direction, whereas the mother had a random pattern. Further studies are necessary to establish a possible association between the expression of WBS and X chromosome inactivation.

Inheritance of skewed X chromosome inactivation in a large family with an X-linked recessive deafness syndrome.

A new X-linked recessive deafness syndrome was recently reported and mapped to Xq22 (Mohr-Tranebjaerg syndrome). In addition to deafness, the patients had visual impairment, dystonia, fractures, and mental deterioration. The female carriers did not have any significant manifestations of the syndrome. We examined X chromosome inactivation in 8 obligate and 12 possible carriers by using a polymerase chain reaction analysis of the methylation-dependent amplification of the polymorphic triplet repeat at the androgen receptor locus. Seven of 8 obligate carriers and 1 of 5 carriers by linkage analysis had an extremely skewed pattern in blood DNA not found in 30 normal females. The X inactivation pattern in fibroblast DNA from 2 of the carriers with the extremely skewed pattern was also skewed but to a lesser degree than in blood DNA. One obligate carrier had a random X inactivation pattern in both blood and fibroblast DNA. A selection mechanism for the skewed pattern is therefore not likely. The extremely skewed X inactivation in 8 females of 3 generations in this family may be caused by a single gene that influences skewing of X chromosome inactivation.

Interaction of Shigella shigae cytotoxin with receptors on sensitive and insensitive cells.

The effect of a cytotoxin isolated from Shigella shigae has been tested on different cell lines. HeLa S3 cells, as well as some other human carcinoma cells, were killed by picomolar to femtomolar concentrations of the pure toxin, whereas certain other human carcinoma cells and a variety of non-epithelial cells from human tissue and from various animal tissues were resistant to-nanomolar concentrations of the toxin. Binding studies with 125 I-labelled Shigella shigae cytotoxin showed that the sensitive HeLa S3 cells contain 1.3 x 10(6) binding sites per cell, whereas in an insensitive HeLa cell line 2.6 x 10(5) sites per cell were measured. In all cases the apparent association constant, Ká was found to be about 10(10) M-1. The binding occurred fairly rapidly, whereas dissociation of bound toxin occurred at a very slow rate, even in the presence of excess unlabaled toxin. All toxin sensitive cell lines bound similar amounts of toxin as HeLa S3 cells, whereas some of the resistant cell lines did not contain measurable amounts of toxin receptors.

Isolation and characterization of Shigella shigae cytotoxin.

Shigella shigae cytotoxin was isolated from pressure-dialyzed culture medium and from a 26-year-old sample of partially purified toxin. The toxin was adsorbed to a column of acid-treated chitin at low salt concentration and eluted with 1 M NaCl. The partially purified toxin was labeled with 125 I and resubmitted to chromatography on acid-treated chitin. The labeled material eluted with 1 M NaCl was mixed with unlabeled rabbit hemoglobin as a carrier and the toxin was further purified by chromatography on a DE52 column and by sucrose gradient centrifugation. In polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the pure Shigella toxin migrated as two bands corresponding to molecular weights of 30,500 and of about 11,000. The intact toxin may consist of one heavy chain and four to five copies of the light chain. In isoelectric focusing experiments, Shigella toxin was recovered from a broad zone between pH 5.8 and pH 7.5. This appears to be due to charge heterogeneities both in the large and the small chain. Most cell lines tested were completely resistant even to high concentrations of Shigella toxin. Vero cells and one strain of HeLa cells were very sensitive, 2.5 pg/ml of pure toxin induced 50% inhibition of protein synthesis overnight in HeLa cells.

Animal toxicity of Shigella dysenteriae cytotoxin: evidence that the neurotoxic, enterotoxic, and cytotoxic activities are due to one toxin.

The lethal effect to rabbits and mice of Shigella dysenteriae toxin and the ability of the toxin to induce fluid accumulation in rabbit ileal loops were studied in relation to the cytotoxic activity. The relative concentrations of the three activities were approximately the same in a crude toxin preparation and in purified, electrophoretically homogenous toxin. The cytotoxic and lethal activities eluted identically from a high pressure liquid chromatography column and migrated at the same rate in polyacrylamide gel electrophoresis under nondenaturing conditions. The cytotoxic, lethal, and enterotoxic activities were inactivated to essentially the same extent upon incubation for few minutes at 80 degrees C and upon treatment with urea. Graded precipitation of Shigella toxin with different amounts of an antiserum to Shigella toxin in each case removed essentially the same fraction of the cytotoxic, the lethal, and the enterotoxic activity. The data indicate that one molecular entity is responsible for the three biologic effects of Shigella toxin studied. After i.v. injection, the LD50 dose was estimated to be 2.2 ng/kg in rabbits and 450 ng/kg in mice.

Entry of Shigella dysenteriae toxin into HeLa cells.

The rate of shigella toxin entry into the cytosol of HeLa S3 cells was estimated from the toxin-induced reduction in protein synthesis. Whereas high toxin concentrations strongly reduced protein synthesis within 30 min, lower concentrations required longer times. The major part of the cell-bound toxin entered only after several hours. Toxin entered cells after incubation at 25 degrees C but not at 20 degrees C, although toxin binding was the same at the two temperatures. Increasing the KCl concentration to 0.2 M protected against toxin. The toxin entry was strongly reduced when the level of ATP in the cells was reduced by incubation with metabolic inhibitors. Lysosomotrophic agents such as NH4Cl and chloroquine had little or no protective effect, but the protonophores carbonyl cyanide p-trifluoromethoxyphenylhydrazone and carbonyl cyanide m-chlorophenylhydrazone and the ionophore monensin protected cells against the toxin. Cells were also protected when the pH was reduced to 6.4. The entry of shigella toxin is discussed in relation to that of other protein toxins with intracellular sites of action.

The cytotoxic activity of Shigella toxin. Evidence for catalytic inactivation of the 60 S ribosomal subunit.

The cytotoxic test system for Shigella shigae toxin was improved and used to study the stability of the toxin to various pH values, temperature, and chemicals. Inhibition of protein synthesis is the first demonstrable effect in cells treated with Shigella toxin. This inhibition appears to be at the level of peptide chain elongation. An inhibition effect on cell-free protein synthesis is exhibited by toxin pretreated first with trypsin and then with dithiothreitol and 8 M urea or 1% sodium dodecyl sulfate. Ribosomes treated with toxin or its A1 fragment had lost most of their ability to polymerize [14C]phenylalanine in a poly(U)-dependent cell-free system. Salt-washed ribosomes in simple buffered solutions were inactivated at a rate of at least 40 ribosomes/(min) (A1 fragment). Addition of antitoxin immediately stopped further inactivation, but it did not reactivate the inactivated ribosomes. 60 S ribosomal subunits from toxin-treated ribosomes had a marked reduction in ability to support polyphenylanine synthesis, whereas 40 S subunits from toxin-treated ribosomes retained their activity. Toxin-treated ribosomes retained their ability to incorporate [3H]puromycin into growing peptide chains, indicating that the peptide bond formation is not the function inhibited.

Carnitine deficiency induced during intermittent haemodialysis for renal failure.

Carnitine concentration was measured in plasma, muscle, and dialysate before and after haemodialysis in patients with renal failure and in plasma and muscle of healthy controls. In eight of the nine patients carnitine concentration in muscle after haemodialysis was only 10% of the concentration in controls. Plasma-carnitine varied in patients before dialysis and in all of them was reduced by dialysis. The loss of carnitine into the dialysate (190--2100 mumol/treatment) greatly exceeded the normal loss in urine for most of patients, and was only partly compensated for. In some patients normal or high plasma-carnitine and low concentrations in muscle indicated that the carnitine-concentrating mechanisms in the muscle cell had failed. The reduction in carnitine will interfere seriously with normal cellular functions and this may help to explain the clinical syndrome of cardiomyopathy and cardiac failure which has been observed in some patients treated for a long time with intermittent haemodialysis.

Subunit structure of Shigella cytotoxin.

Shigella cytotoxin was purified from the culture medium of Shigella shigae 60 R and from the bacterial pellet by an extensive purification scheme involving chromatography on Cibachron Blue F3GA, chitin, and DEAE-cellulose columns, sucrose gradient centrifugation, and finally polyacrylamide gel electrophoresis under nondenaturing conditions. In sodium dodecyl sulfate polyacrylamide gels, purified toxin showed two bands, a heavy one with the molecular weight of 30,500 (A chain), and a broad band migrating near the dye front (B chain). The A chain was easily cleaved by proteases into two fragments, A1 (Mr = 27,500) and A2 (Mr = 3,000). These fragments were linked with a disulfide bridge (nicked toxin). The molecular weight of native toxin was estimated by the method of Ferguson to be 68,000 +/- 4,000. Cross-linking experiments indicated that the native toxin consists of one A chain and six to seven B chains, with each B chain having a molecular weight of congruent to 5,000. The isolated A and B chains were not toxic to cells nor did they bind to cells to a measurable extent. In a cell-free system, the A1 fragment strongly inhibited protein synthesis. The possibility that the B chains form the binding moiety of the toxin is discussed.

Properties and action mechanism of the toxic lectin modeccin: interaction with cell lines resistant to modeccin, abrin, and ricin.

The toxic lectin modeccin, which inhibits protein synthesis in eukaryotic cells, is cleaved upon treatment with 2-mercaptoethanol into two peptide chains which move in polyacrylamide gels at rates corresponding to molecular weights 28,000 and 38,000. After reduction, the toxin loses its effect on cells, while its ability to inhibit cell-free protein synthesis increases. Like abrin and ricin it inhibits protein synthesis by inactivating the 60S ribosomal subunits. Modeccin binds to surface receptors containing terminal galactose residues. Competition experiments with various glycoproteins indicate that the modeccin receptors are different from the abrin receptors. In addition, they were present on HeLa cells in much smaller numbers. Moreover, mutant lines resistant to abrin and ricin were not resistant to modeccin and vice-versa. The toxin resistance of various mutant cell lines could not be accounted for by a reduced number of binding sites on cells. The data are consistent with the view that the cells possess different populations of binding sites with differences in ability to facilitate the uptake of the toxins and that in the resistant lines the most active receptors have been reduced or eliminated.

Hypoplasia of the sphenoid sinuses as a diagnostic tool in cystic fibrosis.

PURPOSE: To measure and compare the size of the sphenoid sinuses in patients with cystic fibrosis (CF) to patients with inflammatory sinonasal disease, and to correlate the size with number of CF mutations in each patient. MATERIAL AND METHODS: Ninety-six CF patients aged 5-47 years (median 19 years) and 130 control patients aged 7-51 years (median 32 years) were examined using coronal CT of the paranasal sinuses. In each patient, the CT image with the largest coronal area of the sphenoid sinuses was scanned into a Macintosh computer with image processing and analysis software. Largest coronal area and largest circumference of the right and left sphenoid sinuses were automatically measured. Additionally, antero-posterior extension of the sphenoid sinuses was calculated from the lateral scanograms. CF patients were grouped according to number of confirmed mutations (CF-0, CF-1, or CF-2). RESULTS: CF patients generally had small sphenoid sinuses. The largest differences for all parameters were observed between the CF-2 and the control groups (p<0.0001). No CF-2 patient had pneumatization beyond the presphenoid. The CF-0 and CF-1 groups consisted of two populations, one overlapping the CF-2 group and another overlapping the control group. CONCLUSION: Hypoplasia of the sphenoid sinuses is a characteristic finding in CF patients. When pneumatization of the basisphenoid is present, the existing CF diagnosis should be questioned.

CT characterization of developmental variations of the paranasal sinuses in cystic fibrosis.

PURPOSE: To describe variations of paranasal sinus development in patients with cystic fibrosis (CF) and in non-CF patients examined for inflammatory sinonasal disease. We focused on anatomic variants that predispose to orbital and cerebral penetration during functional endoscopic sinus surgery (FESS), e.g. hypoplasia of the maxillary sinus and low ethmoid roof. MATERIAL AND METHODS: One hundred and sixteen CF patients (3-54 years, median 18) and 136 control patients (7-51 years, median 31) were examined with coronal CT of the paranasal sinuses. CF patients were grouped according to number of confirmed mutations: CF-2 (n=70), CF-1 (n=32), CF-0 (n=14). CT images were evaluated with respect to paranasal sinus development, pneumatization variants and bony variants. RESULTS: Frontal sinus aplasia and maxillary, ethmoid, and sphenoid sinus hypoplasia were markedly more frequent in CF-2 than in control patients. No CF-2 patient had pneumatization variants such as Haller cells or concha bullosa. Low ethmoid roof was seen in 30% of CF-2 children, but in no control children. CF-1 and CF-0 groups had prevalences of aplasia and hypoplasia intermediate to that of CF-2 and control patients. CONCLUSION: Genetically verified CF patients had less developed sinuses, lacked pneumatization variants, and more often had anatomic variants that predispose to complications during FESS. Normally developed sinuses and pneumatization variants in some genetically unverified CF patients (CF-1, CF-0) suggest that these patients may be erroneously diagnosed.

Proposal of a CT scoring system of the paranasal sinuses in diagnosing cystic fibrosis.

The purpose of this study was to develop a paranasal sinus CT scoring system that could be used as a diagnostic tool to discriminate cystic fibrosis (CF) patients from control patients examined for sinonasal disease. The model should include as few and easily applicable criteria as possible, supported by statistical analyses and clinical judgement. We used data from 116 CF and 136 control patients. The CF patients were grouped according to the number of confirmed CF mutations: genetically verified (CF-2), or based on sweat testing and clinical findings alone (CF-1, CF-0). Nine paranasal sinus CT criteria, including development, pneumatisation variants and inflammatory patterns, were evaluated. The final model included three criteria: (a) frontal and (b) sphenoid sinus development, and (c) absence of three pneumatisation variants. This model discriminated CF-2 from controls with overlap of summed scores in only 8 of 206 patients. When this model was applied in the CF-1 and CF-0 groups, two populations seemed to exist. A larger group with summed scores overlapping that of the CF-2 group and a smaller group with summed scores overlapping that of the control group. We conclude that this CT scoring system may support, as well as exclude, a CF diagnosis in cases of diagnostic uncertainty.

394delTT: a Nordic cystic fibrosis mutation.

In a systematic screening for mutations in the gene encoding the cystic fibrosis transmembrane regulator among Danish cystic fibrosis (CF) patients, we identified a mutation in exon 3 (394delTT); this mutation was found to be relatively common in Denmark. We therefore screened for 394delTT in Sweden and Norway, where it turned out to be the second most frequent mutation, accounting for 4% of all CF mutations. It also occurs with a high frequency in Finland, but has not been found in larger surveys of mutations in the CFTR gene. Thus, 394delTT seems to be a specific Nordic CF mutation.

Frequency of the delta F508 and exon 11 mutations in Norwegian cystic fibrosis patients.

We have searched for the delta F508 mutation in 77 Norwegian cystic fibrosis patients. Of the 154 chromosomes tested, 93 (60%) carried the delta F508 mutation. Haplotypes at the D7S23 locus (KM19 and XV2C markers) were determined. Of 81 chromosomes with the F508 mutation, the B haplotype was found on 77. We found three patients with the G551D and one patient with the R553X mutation in exon 11 of the CFTR locus.

Mapping of the locus for cholestasis-lymphedema syndrome (Aagenaes syndrome) to a 6.6-cM interval on chromosome 15q.

Patients with cholestasis-lymphedema syndrome (CLS) suffer severe neonatal cholestasis that usually lessens during early childhood and becomes episodic; they also develop chronic severe lymphedema. The genetic cause of CLS is unknown. We performed a genome screen, using DNA from eight Norwegian patients with CLS and from seven unaffected relatives, all from an extended pedigree. Regions potentially shared identical by descent in patients were further characterized in a larger set of Norwegian patients. The patients manifest extensive allele and haplotype sharing over the 6.6-cM D15S979-D15S652 region: 30 (83.3%) of 36 chromosomes of affected individuals carry a six-marker haplotype not found on any of the 32 nontransmitted parental chromosomes. All Norwegian patients with CLS are likely homozygous for the same disease mutation, inherited from a shared ancestor.