Aceruloplasminaemia: a family with a novel mutation and long-term therapy with deferasirox.

Ceruloplasmin is a member of the multicopper oxidase family that plays a major role in the transport of iron in the body. Aceruloplasminaemia (ACP) is a rare disease and is clinically identified by iron overload in liver, pancreas, brain, and other organs, and by microcytic anaemia. So far, the iron chelator deferasirox was given for therapy only up to 6 months due to side effects. Here, we describe a novel mutation leading to ACP and report for the first time a long-term therapy, that is, 2 years with deferasirox. ACP was diagnosed in 3 siblings using clinical and biochemical characteristics, HFE and ceruloplasmin mutational analysis, liver biopsy, brain-, liver-, and heart-MRI. For iron depletion, a starting dose of deferasirox 7.5 mg/kg/day was increased to 15 mg/kg/day and maintained at 4-7.5 mg/kg/day with a patient follow-up for 2 years. A novel homozygous mutation of the ceruloplasmin gene on chromosome 3 (3q23-q25, exon 12, G708S) was found. Iron was selectively and successfully removed by long-term therapy with deferasirox, as confirmed by follow-up liver biopsies, normalisation of serum ferritin concentrations, and improved glucose metabolism. Unexpectedly, iron depletion ameliorated anaemia. Low-dose deferasirox is an effective and safe long-term treatment option for patients with ACP.

Loss of wild-type MEN1 gene expression in multiple endocrine neoplasia type 1-associated parathyroid adenoma.

Multiple endocrine neoplasia type 1 (MEN1) is a human hereditary tumor syndrome characterized by the development of endocrine adenomas of the parathyroid, anterior pituitary, and enteropancreatic tissue. Several lines of evidence have implicated the recently identified MEN1 gene located on chromosome 11q13 as a recessive tumor suppressor gene. Here, we analyzed MEN1 wild-type gene expression in tumors from a large MEN1 kindred. A deletion of codons 227-228 (678del6) located in exon 4 was found in tumor and peripheral blood complementary DNA using a simplified single-strand conformational polymorphism (SSCP) approach well suited for clinical MEN1 mutation screening. The identified 678del6 cDNA mutation deletes a potential phosphorylation site (Tyr227) and corresponds to a germ line mutation co-segregating with disease phenotype in this MEN1 family. Loss of heterozygosity analysis by fluorescent microsatellite PCR showed an exclusive loss of the MEN1 wild-type (and retention of the mutated) allele detectable in DNA from microdissected parathyroid and pancreatic, but not in adrenal, adenomas. Our findings confirm the synergism between MEN1 gene mutations and subsequent MEN1 allelic losses in the tumorigenesis of MEN1-associated adenomas.

Genomic structure and functional expression of a human alpha(2)/delta calcium channel subunit gene (CACNA2).

CACNA2 encodes the alpha(2)/delta subunit of the human voltage-gated calcium channels and is located in the candidate region of malignant hyperthermia susceptibility type 3 (MHS3). We determined the structural organization of CACNA2 by isolation of overlapping genomic DNA clones from a human phage library. The gene consists of at least 40 exons, 2 of which are alternatively spliced, spanning more than 150 kb of genomic DNA. Exons range from 21 to 159 bp, and introns range from 98 bp to at least more than 20 kb. We constructed a full-length cDNA and cloned it into a mammalian expression vector. Cotransfection of the CACNA2 cDNA with alpha(1A) and beta(4) cDNA into HEK293 cells led to the expression of Q-type calcium currents. The alpha(2)/delta subunit enhanced the current density 18-fold compared to cells transfected with only alpha(1A) and beta(4) cDNA. The sequence analysis provides the basis for comprehensive mutation screening of CACNA2 for putative MHS3 individuals and patients with other channelopathies.

A reduced K+ current due to a novel mutation in KCNQ2 causes neonatal convulsions.

Benign familial neonatal convulsions (BFNC) is a rare dominantly inherited epileptic syndrome characterized by frequent brief seizures within the first days of life. The disease is caused by mutations in one of two recently identified voltage-gated potassium channel genes, KCNQ2 or KCNQ3. Here, we describe a four-generation BFNC family carrying a novel mutation within the distal, unconserved C-terminal domain of KCNQ2, a 1-bp deletion, 2513delG, in codon 838 predicting substitution of the last seven and extension by another 56 amino acids. Three family members suffering from febrile but not from neonatal convulsions do not carry the mutation, confirming that febrile convulsions and BFNC are of different pathogenesis. Functional expression of the mutant channel in Xenopus oocytes revealed a reduction of the potassium current to 5% of the wild-type current, but the voltage sensitivity and kinetics were not significantly changed. To find out whether the loss of the last seven amino acids or the C-terminal extension because of 2513delG causes the phenotype, a second, artificial mutation was constructed yielding a stop codon at position 838. This truncation increased the potassium current by twofold compared with the wild type, indicating that the pathological extension produces the phenotype, and suggesting an important role of the distal, unconserved C-terminal domain of this channel. Our results indicate that BFNC is caused by a decreased potassium current impairing repolarization of the neuronal cell membrane, which results in hyperexcitability of the central nervous system.

Screening of the ryanodine receptor gene in 105 malignant hyperthermia families: novel mutations and concordance with the in vitro contracture test.

Malignant hyperthermia (MH) in man is an autosomal dominant disorder of skeletal muscle Ca(2+)-regulation. During anesthesia in predisposed individuals, it is triggered by volatile anesthetics and depolarizing muscle relaxants. In >50% of the families, MH susceptibility is linked to the gene encoding the skeletal muscle ryanodine receptor (RYR1), the calcium release channel of the sarcoplasmic reticulum, on chromosome 19q12-13.2. To date, 21 RYR1 mutations have been identified in a number of pedigrees. Four of them are also associated with central core disease (CCD), a congenital myopathy. Screening for these 21 mutations in 105 MH families including 10 CCD families phenotyped by the in vitro contracture test (IVCT) according to the European protocol revealed the following approximate distribution: 9% Arg-614-Cys, 1% Arg-614-Leu, 1% Arg-2163-Cys, 1% Val-2168-Met, 3% Thr-2206-Met and 7% Gly-2434-Arg. In one CCD family, the disease was caused by a recently reported MH mutation, Arg-2454-His. Two novel mutations, Thr-2206-Arg and Arg-2454-Cys were detected, each in a single pedigree. In the 109 individuals of the 25 families with RYR1 mutations cosegregation between genetic result and IVCT was almost perfect, only three genotypes were discordant with the IVCT phenotypes, suggesting a true sensitivity of 98.5% and a specificity of minimally 81.8% for this test. Screening of the transmembraneous region of RYR1 did not yield a new mutation confirming the cytosolic portion of the protein to be of main functional importance for disease pathogenesis.

Transient weakness and compound muscle action potential decrement in myotonia congenita.

Twenty-five Turkish patients with recessive myotonia congenita (RMC), 16 of whom had genetic confirmation, were studied. Nineteen had transient weakness. In the upper extremities, onset age of transient weakness was usually in the early teens. All untreated RMC patients had a compound muscle action potential decrement of > or =25%, usually above 50%, with repetitive nerve stimulation at 10/s for 5 s. Patients with other nondystrophic diseases with myotonia, except 1 patient with dominant myotonia congenita, had no transient weakness and a CMAP decrement below 25%.

The dominant chloride channel mutant G200R causing fluctuating myotonia: clinical findings, electrophysiology, and channel pathology.

Clinical, electrophysiological, and molecular findings are reported for a family with dominant myotonia congenita in which all affected members have experienced long-term fluctuations of the symptom of myotonia. In some patients myotonia is combined with myalgia. The myotonia-causing mutation in this family is in the gene encoding the muscular chloride channel, hCIC-1, predicting the amino acid exchange G200R. We have constructed recombinant DNA vectors for expression of the mutant protein in tsA201 cells and investigation of the properties of the mutant channel. The most prominent alteration was a +100-mV shift of the midpoint of the activation curve. Therefore, within the physiological range the open probability of the mutant channel is markedly smaller than in wild-type. This shift is likely to be responsible for the myotonia in the patients. The fluctuating symptoms of this chloride channelopathy are discussed with respect to short-term fluctuations of myotonia in the sodium channelopathy of potassium-aggravated myotonia.

An oncogenic fusion product of the phosphatidylinositol 3-kinase p85beta subunit and HUMORF8, a putative deubiquitinating enzyme.

Peripheral blood cell DNA from a patient with a chronic myeloproliferative disorder was tested in the tumorigenicity assay. Upon tumor induction in nude mice we isolated a human oncogene by means of genomic cloning, exon trap analysis and cDNA cloning. Sequence analysis revealed a fusion product of the p85beta subunit of phosphatidylinositol (PI) 3-kinase and HUMORF8, a putative deubiquitinating enzyme, which has been generated during the DNA transfection process. Application of the tumorigenicity assay to various p85beta and HUMORF8 cDNA constructs indicated that the recombination of both genes rather than the truncation of one of the fusion partners renders the chimeric protein tumorigenic. Moreover, sequence analysis of human wildtype p85beta revealed an alanine for serine substitution at a site important for the regulation of the lipid kinase activity of PI 3-kinase in human p85alpha. This variation may relate to differences in the mode of signal transduction from both p85 isoforms.

Intracellular signaling of the Ufo/Axl receptor tyrosine kinase is mediated mainly by a multi-substrate docking-site.

Ufo/Axl belongs to a new family of receptor tyrosine kinases with an extracellular structure similar to that of neural cell adhesion molecules. In order to elucidate intracellular signaling, the cytoplasmic moiety of Ufo/Axl was used to screen an expression library according to the CORT (cloning of receptor targets) method. Three putative Ufo substrates were identified: phospholipase Cgamma1 (PLCgamma), as well as p85alpha and p85beta subunits of phosphatidylinositol 3'-kinase (PI3-kinase). Subsequently, chimeric EGFR/Ufo receptors consisting of the extracellular domains of the epidermal growth factor receptor (EGFR) and the transmembrane and intracellular moiety of Ufo were engineered. Using different far-Western blot analyses and coimmunoprecipitation assays, receptor binding of PLCgamma and p85 proteins as well as GRB2, c-src and lck was examined in vitro and in vivo. Competitive inhibition of substrate binding and mutagenesis experiments with EGFR/Ufo constructs revealed C-terminal tyrosine 821 (EILpYVNMDEG) as a docking site for multiple effectors, namely PLCgamma, p85 proteins, GRB2, c-src and lck. Tyrosine 779 (DGLpYALMSRC) demonstrated an additional, but lower binding affinity for the p85 proteins in vitro. In addition, binding of PLCgamma occurred through tyrosine 866 (AGRpYVLCPST). Moreover, our in vivo data indicate that further direct or indirect binding sites for PLCgamma, GRB2, c-src and lck on the human Ufo receptor may exist.

Functional characterization of a distinct ryanodine receptor mutation in human malignant hyperthermia-susceptible muscle.

Malignant hyperthermia is an inherited autosomal disorder of skeletal muscle in which certain volatile anesthetics and depolarizing muscle relaxants trigger an abnormally high release of Ca2+ from the intracellular Ca2+ store, the sarcoplasmic reticulum. In about 50% of cases, malignant hyperthermia susceptibility is linked to the gene encoding the skeletal muscle ryanodine receptor/Ca2+ release channel (RYR1). To date, eight point mutations have been identified in human RYR1. Although these mutations are thought to lead to an increased caffeine and halothane sensitivity in the contractile response of skeletal muscle, their functional consequences have not been investigated on the molecular level. In the present study, we provide the first functional characterization of a point mutation located in the central part of RYR1, Gly2434 --> Arg. Using high affinity [3H]ryanodine binding as the experimental approach, we show that this mutation enhances the sensitivity of RYR1 to activating concentrations of Ca2+ and to the exogenous and diagnostically used ligands caffeine and 4-chloro-m-cresol. In parallel, the sensitivity to inhibiting concentrations of Ca2+ and calmodulin was reduced, transferring the mutant Ca2+ release channel into a hyperexcitable state.

Multiparameter approach in the identification of cross-contaminated leukemia cell lines.

A common problem in cell culturing is cross-contamination with other cells or misidentification of cells. An effective cell culture quality and identity control is required in order to avoid inter- and intraspecies contamination of cell lines and their further propagation and dissemination. We present evidence that supposedly unrelated cell lines that we received from the original investigators are in fact related to the chronic myeloid leukemia cell line K-562. The sister cell lines SPI-801 and SPI-802 were originally established from a patient with T-cell acute lymphoblastic leukemia and displayed T-cell associated features. However, data from morphological evaluation, immunophenotyping, bcr-abl gene rearrangement analysis, DNA fingerprinting, Northern blot analysis of globin gene expression and esterase isoenzyme analysis clearly established that the three cell lines are related. Cytogenetic examination while not proving the common identity of the cells provided further evidence for the suspected common origin of all three cell lines. Chromosome banding, DNA fingerprinting and bcr-abl genotyping suggested further evolution of these clones during long-term cultivation. Quality and identity control is an essential feature of cell culture technique. Only regular monitoring for purity and integrity of cell lines will significantly reduce the incidence of cell line contamination and misidentification.

The genomic structure of the human UFO receptor.

Using a DNA transfection-tumorigenicity assay we have recently identified the UFO oncogene. It encodes a tyrosine kinase receptor characterized by the juxtaposition of two immunoglobulin-like and two fibronectin type III repeats in its extracellular domain. Here we describe the genomic organization of the human UFO locus. The UFO receptor is encoded by 20 exons that are distributed over a region of 44 kb. Different isoforms of UFO mRNA are generated by alternative splicing of exon 10 and differential usage of two imperfect polyadenylation sites resulting in the presence or absence of 1.5-kb 3' untranslated sequences. Primer extension and S1 nuclease analyses revealed multiple transcriptional initiation sites including a major site 169 bp upstream of the translation start site. The promoter region is GC rich, lacks TATA and CAAT boxes, but contains potential recognition sites for a variety of trans-acting factors, including Sp1, AP-2 and the cyclic AMP response element-binding protein. Proto-UFO and its oncogenic counterpart exhibit identical cDNA and promoter regions sequences. Possible modes of UFO activation are discussed.

Differences in DNA fingerprints of continuous leukemia-lymphoma cell lines from different sources.

The genetic stability of human cell lines in long-term culture has been tested by DNA fingerprinting a panel of 31 different continuous cell lines from patients with leukemias or lymphomas. Duplicates of the same cell line obtained from different sources, subclones of cell lines, and samples of cell lines at different passage levels were studied. In most cases the fingerprints of duplicates of the same cell line remained perfectly preserved even after long-time passaging. However, in five cases there were notable differences between individual fragments of corresponding fingerprints. We have found four cases of mislabeled and/or cross-contaminated cell lines so far. Taken together, our results indicate that DNA fingerprinting qualifies as a very reliable means of cell line identification which allows the detection of mislabelling or contamination and of genetic variation among subclones.

The murine ufo receptor: molecular cloning, chromosomal localization and in situ expression analysis.

We have cloned the mouse homologue of the ufo oncogene. It encodes a novel tyrosine kinase receptor characterized by a unique extracellular domain containing two immunoglobulin-like and two fibronectin type III repeats. Comparison of the predicted ufo amino acid sequences of mouse and man revealed an overall identity of 87.6%. The ufo locus maps to mouse chromosome 7A3-B1 and thereby extends the known conserved linkage group between mouse chromosome 7 and human chromosome 19. RNA in situ hybridization analysis established the onset of specific ufo expression in the late embryogenesis at day 12.5 post coitum (p.c.) and localized ufo transcription to distinct substructures of a broad spectrum of developing tissues (e.g. subepidermal cells of the skin, mesenchymal cells of the periosteum). In adult animals ufo is expressed in cells forming organ capsules as well as in connective tissue structures. ufo may function as a signal transducer between specific cell types of mesodermal origin.