Inhibition of 7-dehydrocholesterol reductase by the teratogen AY9944: a rat model for Smith-Lemli-Opitz syndrome

Our aim is to verify the validity of a rat model proposed for Smith-Lemli-Opitz (SLO) syndrome, a developmental disorder characterized by a defect in 7-dehydrocholesterol-delta 7 (7DHC)-reductase and by facial dysmorphism close to the holoprosencephaly caused by the teratogen AY9944. We investigated the sterol profile in rats treated with AY9944 blocking 7DHC-reductase. AY9944 was given orally to rats on gestation day 3 (D3). The sera were sampled for kinetic data on D3, D6, D9, D12, and D21. Cholesterol was measured in parallel by the routine enzymatic method and by the gas chromatography/mass spectrometry (GC-MS) procedure used in SLO diagnosis. In addition to sterols, we dosed steroid hormones punctually on D4 and on D10, and examined D21 fetuses in other animals. The enzymatic method was not specific for cholesterol, and measured 70% pure 7DHC added to a normal serum. On D21, 77% live fetuses showed pituitary agenesis. Cholesterol was rapidly reduced by more than 50% on D6 involving an accumulation of 7DHC, 8DHC, and trienols, as identified in SLO-affected children. The most abundant 7DHC reached a maximum from D9 to D12, equaling cholesterol on D9 (11 mg/dl). On D10, the magnitudes of hypocholesterolemic and of 7DHC accumulation were found to be dose-dependent. Progesterone was reduced as early as 24 hr after treatment and dropped to 40% of the levels in the controls on D10, correlating to the decrease in cholesterolemia. This rat model reproduces the same biochemical perturbations as seen in SLO, strongly suggesting that aberrant sterols (7DHC, 8DHC, or nortrienol) may contribute to the developmental defects.     

Prenatal diagnosis of Smith-Lemli-Opitz syndrome is possible by measurement of 7-dehydrocholesterol in amniotic fluid

Amniocentesis was performed at 17.3 weeks in a pregnancy with severe intrauterine growth retardation. Cytogenetic studies on amniocytes were normal, 46,XX, and the pregnancy was continued. The diagnosis of Smith-Lemli-Opitz syndrome was suspected in the neonatal period and confirmed by the presence of 7-dehydrocholesterol (7-DHC) in the plasma (0.4 mmol/l, normal = not detectable) associated with a low total cholesterol concentration (0.4 mmol/l, normal = 2.56 +/- 0.23). Retrospective analysis of the amniotic fluid sample revealed an elevated level of 7-DHC (0.022 mmol/l; normal = undetectable). Therefore measurement of 7-DHC levels in amniotic fluid during the second trimester of pregnancy is useful for the prenatal diagnosis of Smith-Lemli-Opitz syndrome in families at risk and should be considered in cases of severe growth retardation of unknown aetiology for which amniotic fluid is available and in which a normal chromosomal pattern in amniocytes is present.     

Mutations in the Delta7-sterol reductase gene in patients with the Smith-Lemli-Opitz syndrome

The Smith-Lemli-Opitz syndrome (SLOS) is an inborn disorder of sterol metabolism with characteristic congenital malformations and dysmorphias. All patients suffer from mental retardation. Here we identify the SLOS gene as a Delta7-sterol reductase (DHCR7, EC 1.3.1. 21) required for the de novo biosynthesis of cholesterol. The human and murine genes were characterized and assigned to syntenic regions on chromosomes 11q13 and 7F5 by fluorescense in situ hybridization. Among the mutations found in patients with the SLOS, are missense (P51S, T93M, L99P, L157P, A247V, V326L, R352W, C380S, R404C, and G410S), nonsense (W151X), and splice site (IVS8-1G>C) mutations as well as an out of frame deletion (720-735 del). The missense mutations L99P, V326L, R352W, R404C, and G410S reduced heterologous protein expression by >90%. Our results strongly suggest that defects in the DHCR7 gene cause the SLOS.     

Markedly increased tissue concentrations of 7-dehydrocholesterol combined with low levels of cholesterol are characteristic of the Smith-Lemli-Opitz syndrome

The Smith-Lemli-Opitz syndrome is an autosomal recessive birth defect (frequency 1:20,000-1:40,000) that results in profound mental retardation, physical deformities, and failure to thrive. It is characterized biochemically by low plasma cholesterol and greatly elevated levels of two dehydrocholesterols, one of which is the cholesterol precursor 7-dehydrocholesterol. To determine whether the block in cholesterol biosynthesis affects tissue sterols, we assayed several organs from two affected individuals, a female who died at 27 hours and a 20-week male fetus. Cholesterol concentrations in abdominal wall, adrenal gland, and kidney from two or three unaffected fetuses, who served as controls, averaged 2.0, 1.5, and 1.4 mg/g wet weight, compared to 0.08, 0.44, and 0.14, respectively, for the homozygous fetus. Cerebral cortex cholesterol concentrations were 2.2 mg/g for two 20-22-week fetal controls but only 0.21 and 0.09 mg/g, respectively, for the homozygous child and fetus. Similarly, tissue cholesterol levels were abnormally low in the homozygous child being less than 1 mg/g in liver, adipose, thymus, muscle, and adrenal and 6.2 mg/dl in plasma. Dehydrocholesterols could not be detected by conventional means in any controls but were elevated enough in tissues from affected individuals to make total sterol concentrations nearly normal. These results suggest that a defect in 3 beta-hydroxysterol delta 7-reductase leads to both a profound lack of cholesterol and its replacement by dehydrocholesterols. Such a combination may be lethal in the most severely affected individuals.     

Abnormal cholesterol biosynthesis in the Smith-Lemli-Opitz syndrome

The Smith-Lemli-Opitz syndrome is caused by an inherited defect in 7-dehydrocholesterol-delta7-reductase, the enzyme that catalyzes the last reaction in cholesterol biosynthesis, the conversion of 7-dehydrocholesterol to cholesterol. As a result, deficient cholesterol is produced and the precursor 7-dehydrocholesterol and derivatives (8-dehydrocholesterol and 19-nor-5,7,9(10)-cholestatrien-3 beta-ol) accumulate. Tissues (especially brain) deprived of cholesterol, or because of the deposited sterol precursors and derivatives, develop abnormally and function poorly. Replacement with dietary cholesterol may help correct the biochemical defects and improve symptoms.     

Three cases of Gitelman's syndrome possibly caused by different mutations in the thiazide-sensitive Na-Cl cotransporter

Three adult Japanese cases of Gitelman's syndrome were characterized by secondary aldosteronism, hypokalemic alkalosis, hypomagnesemia, and hypocalciuria. Two were revealed to be familial cases. A mutation in the thiazide-sensitive Na-Cl cotransporter gene, which had already been confirmed in one family (Takeuchi et al. J Clin Endocrinol Metab 81: 4496,1996), was not detected in the other two cases. These observations may possibly support the previous report (Simon et al. Nature Genet 12: 24, 1996) that Gitelman's syndrome is caused by a variety of mutations in the thiazide-sensitive Na-Cl cotransporter.     

Thoracic outlet compression syndrome caused by a schwannoma of the C7 nerve root

This is the first report of a schwannoma originating from the C7 nerve root causing thoracic outlet compression syndrome. The patient was a 30-year-old woman with a 3-year history of numbness on the radial side of the left hand, left arm tiredness, nocturnal pain in the left forearm and pain in the left elbow, shoulder and neck. Conservative treatment and previous operations, including carpal tunnel release and first rib resection, provided no relief. A left scalenectomy was performed. During the removal of the anterior scalene muscle, a mass approximately 3 cm long and 1.5 cm in diameter was noted under the anterior scalene muscle involving the C7 nerve root. The tumour was encapsulated and covered with attenuated and stretched nerve fascicles. It was completely excised without disturbing the nerve fascicles. The clinical impression was schwannoma, which was confirmed on pathological examination.     

Atelosteogenesis type II is caused by mutations in the diastrophic dysplasia sulfate-transporter gene (DTDST): evidence for a phenotypic series involving three chondrodysplasias

Flow cytometric DNA analysis was performed in combination with three-colour immunological staining of cell surface antigens on density-separated mononuclear cells (MNC) obtained from peripheral blood (PB) before, during and after cytokine stimulation of healthy adults. The aim of the study was to determine the cell-cycling status of haemopoietic progenitor cells mobilized into the blood of healthy volunteers during a 5 d treatment period with 5/micrograms per kg body weight of either granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-simulating factor (GM-CSF). Despite considerably increasing numbers of CD34+ PB MNC, the latter were not found to be in S/G2M phase, whereas, among the CD34- MNC, the proportion of cells in S/G2M phase increased from < 0.1% to 0.75 +/- 0.4% (GM-CSF) and to 1.34 +/- 0.75% (G-CSF) and dropped again after discontinuation of the cytokine stimulation. These cells expressed CD33 but were negative for CD45RA, CD3, CD19 and CD14 and were thus considered granulopoietic cells. Analogous results were obtained from analyses of cord blood (CB). In contrast, CD34+ cells from bone marrow (BM) were partially (between 9% and 15%) found to be in S/G2M phase. The non-cycling status of PB and progenitor cells was confirmed by the analysis of CD34+ cells enriched from the two cells sources. However, in vitro stimulation of these progenitor cells using IL3, GM-CSF, erythropoietin and steel factor (SF) revealed that, after 48 h in suspension culture, up to 30% of the CD34+ cells were in S/G2m phase. The fact that cycling CD34+ cells are only detectable in BM but not in PB or CB may suggest different adhesive properties of migrating/mobilized 'stem cells' which may require the BM micro-environment for adequate proliferation in vivo.     

Spectrum of mutations in the OCRL1 gene in the Lowe oculocerebrorenal syndrome

The oculocerebrorenal syndrome of Lowe (OCRL) is a multisystem disorder characterized by congenital cataracts, mental retardation, and renal Fanconi syndrome. The OCRL1 gene, which, when mutated, is responsible for OCRL, encodes a 105-kD Golgi protein with phosphatidylinositol (4,5)bisphosphate (PtdIn[4,5]P2) 5-phosphatase activity. We have examined the OCRL1 gene in 12 independent patients with OCRL and have found 11 different mutations. Six were nonsense mutations, and one a deletion of one or two nucleotides that leads to frameshift and premature termination. In one, a 1.2-kb genomic deletion of exon 14 was identified. In four others, missense mutations or the deletion of a single codon were found to involve amino acid residues known to be highly conserved among proteins with PtdIns(4,5)P2 5-phosphatase activity. All patients had markedly reduced PtdIns(4,5)P2 5-phosphatase activity in their fibroblasts, whereas the ocrl1 protein was detectable by immunoblotting in some patients with either missense mutations or a codon deletion but was not detectable in those with premature termination mutations. These results confirm and extend our previous observation that the OCRL phenotype results from loss of function of the ocrl1 protein and that mutations are generally heterogeneous. Missense mutations that abolish enzyme activity but not expression of the protein will be useful for studying structure-function relationships in PtdIns(4,5)P2 5-phosphatases.     

De-novo COL4A5 gene mutations in Alport's syndrome

Before the advent of direct molecular gene analysis the diagnosis of Alport syndrome was operationally based on three of the four classical clinical criteria. Recently, mutations have been identified in the COL4A5 gene, which is involved in X-linked Alport syndrome. Here we describe two de-novo mutations in two unrelated children, a male and a female, both with early onset of the nephropathy, but with only one of the diagnostic criteria, i.e. electron-microscopy alterations. Because of the significant estimated proportion of de-novo mutations this diagnosis should be considered in children with early signs of nephropathy, even without a suggestive family history or clinical picture (ocular or audiologic abnormalities). In the future the diagnosis of Alport syndrome will probably be made on the basis of both clinical findings and molecular analysis. Now Alport syndrome is clearly underdiagnosed.     

Novel mutations identified in exon 7 of phenylalanine hydroxylase gene in Chinese

Exon 7 of the phenylalanine hydroxylase (PAH) gene was analyzed in 45 children affected with classic phenylketonuria (PKU) from northern China by using PCR-single strand conformation polymorphism (PCR-SSCP) technique and DNA direct sequencing. Six missense mutations (i.e. R243Q, R241H, G247V, L249H, P254I and G257V) and one silent mutation (V245V) were identified. The latter three missense mutations were demonstrated as novel mutations in comparison with the PAH mutation Database. One missense mutation (R241H) was first documented in Chinese. Our results showed population and regional differences in the PAH mutation distribution and suggest that there is more than one founding population for PKU in China. The finding of novel mutations will enhance our capability in molecular diagnosis of PKU.     

Human hypertension caused by mutations in the 11 beta-hydroxysteroid dehydrogenase gene: a molecular analysis of apparent mineralocorticoid excess

CONVERSION OF CORTISOL TO CORTISONE: 11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD) is a microsomal enzyme complex which, in humans, catalyses the interconversion between biologically active cortisol and inactive cortisone. This prereceptor signalling mechanism is essential for maintaining the aldosterone selectivity of the intrinsically non-specific mineralocorticoid receptor and for modulating glucocorticoid access to the glucocorticoid receptor. Apparent mineralocorticoid excess (AME) is a syndrome of severe low-renin mineralocorticoid hypertension associated with marked hypokalaemia which arises from a congenital deficiency of 11 beta-HSD. In AME patients, therefore, it is cortisol and not aldosterone which behaves as a potent mineralocorticoid. ISOFORMS OF 11 BETA-HSD: Two isoforms of human 11 beta-HSD have now been characterized and cloned. The type 1 isoform (11 beta-HSD1) is a low-affinity reduced nicotinamide adenine dinucleotide phosphate (NADP) dependent dehydrogenase-oxoreductase which is expressed in predominantly glucocorticoid target tissues and the encoding sequence of which is normal in patients with AME. In contrast, the type 2 isoform (11 beta-HSD2) is a high-affinity NADP-dependent unidirectional dehydrogenase which is expressed in placenta and mineralocorticoid target tissues such as renal collecting ducts and distal colonic epithelia. Exon- and intron-specific polymerase chain reaction amplification of the 11 beta-HSD2 gene from genomic DNA from members of a consanguinous kindred with AME consistently revealed a single missense mutation (C1228T) in two affected sibs and twin stillbirths. This mutation in codon 374 of exon 5 of the 11 beta-HSD2 gene creates an inframe premature stop (TGA) and, as such, results in a truncated 11 beta-HSD2 protein lacking the carboxyl-terminal proline-rich 32 amino acids. In keeping with an autosomal recessive mode of inheritance, both parents were phenotypically and biochemically normal but were heterozygous for this mutation. Unique to this kindred were expression analyses of the native mutant 11 beta-HSD2 enzyme in the stillbirth-affected placenta, which was almost completely devoid of NADP-dependent 11 beta-dehydrogenase activity. Immunohistochemical and Western blot analyses revealed the absence of 11 beta-HSD2 protein using antisera raised against synthetic peptide sequences corresponding either to the carboxyl terminus or other domains of the enzyme. MISSENSE MUTATION: In this kindred with AME, congenital deficiency of 11 beta-HSD activity is due to a single missense mutation in exon 5 of the 11 beta-HSD2 gene. Simultaneous studies by two other groups have similarly revealed no gross deletions or rearrangements of the 11 beta-HSD2 gene, but have described a number of single point mutations and oligonucleotide deletions in exons 3, 4 and 5, and adjacent to a splice site in intron 3. Recombinant expression analysis of site-directed mutant 11 beta-HSD2 complementary DNA constructs suggests a correlation between the predicted severity of these mutations and the biochemical and clinical phenotype. AME AS A CAUSE OF HYPERTENSION: The mutations in the 11 beta-HSD2 gene, together with those currently being sought by us for other kindreds with AME, establishes AME as a monogenic cause of human hypertension and will provide insight into the structure-function relationships of this important enzyme.     

Evidence that trypanothione reductase is an essential enzyme in Leishmania by targeted replacement of the tryA gene locus

Trypanothione reductase (TR), a flavoprotein oxidoreductase central to the unique thiol-redox system that operates in trypanosomatid protozoa, has been proposed as a potential target for the chemotherapy of trypanosomatid infections. In this study, targeted gene replacement was used to obtain evidence that TR is an essential cellular component and that its physiological function is crucial for parasite survival. Precise replacement of the Leishmania donovani tryA gene encoding TR was only possible upon simultaneous expression of the tryA coding region from an episome; in its absence, attempted removal of the last tryA allele invariably led to the generation of an extra copy of tryA, seemingly as a result of selective chromosomal polysomy. Partial replacement mutants were drastically affected in their ability to survive inside cytokine-activated macrophages in a murine model of Leishmania infection. As no compensatory mechanism for the partial loss of TR activity was observed in these mutants and as it was not possible to obtain viable Leishmania devoid of TR catalytic activity, specific inhibitors of this enzyme are likely to be useful anti-leishmanial agents for chemotherapeutic use.     

Nonsense mutations in the OCRL-1 gene in patients with the oculocerebrorenal syndrome of Lowe

A candidate gene, OCRL-1, for the oculocerebrorenal syndrome of Lowe (OCRL) has been identified via positional cloning strategies. We have now developed RT-PCR techniques which allow amplification of nearly all of the open reading frame from total RNA and have used the PCR products for mutational analysis. Single strand conformational polymorphism analysis detected aberrant migration in two unrelated patients, both of whom were shown to have the same nonsense mutation at base 2746 on direct sequencing. An additional patient was found to be missing a segment from his RNA that corresponds to an entire exon. The identification of mutations in the OCRL-1 gene provides strong genetic evidence for its being the gene involved in Lowe syndrome.     

MHC class II gene silencing in trophoblast cells is caused by inhibition of CIITA expression

A. baumannii is a multiresistant bacteria which is recognised as responsible for nosocomial infections and hospital outbreaks. The control of these outbreaks depends on the strain's typing and on the fight's policy against nosocomial infections. An outbreak of A. baumannii is occurred to patients who were hospitalized in Centre Hospitalier de Versailles. To investigate this outbreak, we have determined the biotype (Bouvet's method), the succeptibility pattern (disk diffusion and agar dilution results were analysed with the hierarchical classification and main component analysis) and the total DNA macrorestriction pattern (Pulse Field Gel Electrophoresis using SmaI restriction enzyme). A risk factors for A. baumannii acquisition were delineated in case-control study. During 2 years, 38 patients have been infected or colonized to A. baumannii. Thirty two patients were hospitalized in ICU. We studied 38 non repetitive clinical isolates and 9 strains of the patient's rooms. Four biotypes were defined by the Bouvet's typing method. Fourteen groups were obtained when succeptibility results were analysed with the hierarchical classification and 6 with the main composant analysis. The molecular typing permit us to define 4 epidemic and 6 sporadic strains. All the epidemic strains were isolated on ICU hospitalized patients. Our study has shown wide contamination in patient's rooms (Water tap, dry surfaces, patient's mattresses...). Environmental objects have been a major risk factor for A. baumannii acquisition. The control of this outbreak has been possible by application of hygienic measures (hands washing, isolment, meticulous cleaning of the ICU and environmental controls). No new case is occurred in the last year. Typing methods and case-control study are necessary to investigate cross-infections and take efficient measures against these outbreaks.     

Clinical similarities of hereditary progressive/dopa responsive dystonia caused by different types of mutations in the GTP cyclohydrolase I gene

OBJECTIVE: Hereditary progressive dystonia with pronounced diurnal fluctuation [(HPD)/dopa responsive dystonia (DRD)] is a childhood onset dystonia which responds to levodopa. Various clinical signs and symptoms of HPD/DRD have been recognised to date. Mutations in the GTP cyclohydrolase I (GTP-CH-I) gene were recently identified as the cause of HPD/ DRD. In the present study, the GTP-CH-I gene and the clinical features of eight HPD/DRD patients from six families were analysed to determine the correlations between clinical expression and the mutations in the GTP-CH-I gene. METHODS: The exons, exon-intron junctions, and an indispensable part of the 5' flanking region of the GTP-CH-I gene were sequenced in the eight clinically diagnosed patients with HPD/DRD and their asymptomatic parents. RESULTS: Three independent mutations in the GTP-CH-I gene were found in three patients. One of the patients and her asymptomatic mother were heterozygous for a novel mutation at the initiation codon. The three patients with dissimilar GTP-CH-I mutations exhibited similar clinical features. The other five patients with normal sequences presented several features not manifested by the three patients with the mutations. No mutation was found in the 5' flanking region of any patients or their parents. CONCLUSIONS: A novel initiation codon mutation was found in a Japanese patient with HPD/DRD. The clinical manifestations common to the patients with HPD/ DRD with a mutated GTP-CH-I gene were also identified. Although focal manifestations of HPD/DRD associated with the mutations of this gene will be broadened, it is inferred that these clinical features are fundamental to HPD/DRD caused by mutations in this gene.     

The thrombocytopenia syndrome caused by heparin

Heparin represents the basis for drug treatment and prevention of thrombosis. However, heparin itself may produce side effects, among others two types of heparin-induced thrombocytopenia (HIT). The more frequent type 1 occurs in about 10% of patients within the first days of the treatment and disappears spontaneously without sequelae. HIT type 2 represents a far more serious complication occurring in 0.1-1% of patients somewhat later, between days 4 and 10 of heparin therapy, and may provoke severe arterial and venous thrombosis. Apart from the differences in clinical picture, data regarding platelet count and platelet aggregation test are significant for the diagnosis. Heparin should be discontinued immediately and another anticoagulant therapy introduced. We are presenting the case of a patient with thrombosis of the left external iliac artery following surgery for ovarian cancer as a complication of HIT type 2, which was recanalized successfully and the leg was saved.