[Molecular biology and pathology of cystic fibrosis. Two years after cloning of the gene].
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Child↗
Biomedical subjects
M Goossens
Publications and source records attributed to M Goossens.
Defective membrane expression of human growth hormone (GH) receptor causes Laron-type GH insensitivity syndrome.
Mutations in the growth hormone receptor (GHR) gene can cause growth hormone (GH) resistance. Given the sequence homology between the extracellular domain of the GHR and a soluble GH-binding protein (GH-BP), it is remarkable that GH-BP binding activity is absent from the serum of patients with Laron-type GH insensitivity, a hereditary form of severe dwarfism. We have previously identified a mutation within the extracellular domain of this receptor, replacing phenylalanine by serine at position 96 of the mature protein, in a patient with Laron syndrome. We have now investigated the effect of this Phe----Ser substitution on hormone binding activity by expressing the total human GHR cDNA and mutant form in eukaryotic cells. The wild-type protein expressed was able to bind GH but no plasma membrane binding was detectable on cells transfected with the mutant cDNA; this was also the case of cells transfected with a Phe96----Ala mutant cDNA, suggesting that the lack of binding activity is not due to a posttranslational modification of serine. Examination of the variant proteins in subcellular fractions revealed the presence of specific GH binding activity in the lysosomal fraction, whereas immunofluorescence studies located mutant proteins in the cytosol. Our findings suggest that these mutant GHRs fail to follow the correct intracellular transport pathway and underline the potential importance of this phenylalanine residue, which is conserved among the GH, prolactin, and erythropoietin receptors that belong to the same cytokine receptor superfamily.
Amino Acid Sequence↗
Haemophilia B: database of point mutations and short additions and deletions--second edition.
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Base Sequence↗
[Amplification of deoxyribonucleic acid sequences].
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Base Sequence↗
Progress in the DNA diagnosis of hemophilias.
DNA diagnosis of hemophilias has received much attention in recent years, and different methods are presently available to analyze the coagulation factor IX and VIII genes. This review is intended to serve as a brief guide to current and emerging diagnostic strategies.
DNA↗
Structure of the Echinococcus multilocularis U1 snRNA gene repeat.
The gene encoding U1 snRNA in Echinococcus multilocularis has been cloned and sequenced. This gene is contained within a 1300-bp sequence which is tandemly repeated in the E. multilocularis genome. E. multilocularis U1 snRNA is 50-70% homologous to U1 snRNAs of other species. E. multilocularis U1 snRNA could assume a predicted secondary structure similar to that proposed for other U1 snRNAs, and appears shorter (157 bases) than the U1 snRNAs of higher eukaryotes (163-166 bases).
Amino Acid Sequence↗
Molecular defects in the growth hormone receptor.
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Cloning, Molecular↗
Identification of a new mutation responsible for hepatoerythropoietic porphyria.
A deficiency in the activity of uroporphyrinogen decarboxylase (URO-D), the fifth enzyme of the haem biosynthetic pathway, is found in two hereditary diseases, familial porphyria cutanea tarda (PCT) and hepatoerythropoietic porphyria (HEP). Little is known about the genetic relationship between those two diseases and it has been postulated that HEP is the homozygous form of PCT. A URO-D cDNA was cloned from an HEP patient and the comparison between the mutant and the wild-type sequences showed a single base difference within the coding sequence leading to the replacement of a glutamic acid by a lysine at codon 167 of the mutant protein. This replacement produced a protein which is rapidly degraded in the presence of cell lysate. On the basis of hybridization of synthetic oligomers to amplified genomic DNA, we demonstrated that this patient is homozygous for this single base mutation. In order to look for any relationship between HEP and PCT, we tested six unrelated patients with familial PCT and could not detect the codon 167 mutation in any of them. These results indicate an heterogeneity in the mutations responsible for the PCT and HEP phenotypes.
Amino Acid Sequence↗
Long-lasting catch-up growth under bio-methionyl growth hormone treatment in an infant with isolated growth hormone deficiency type 1A.
This case report concerns a 7-month-old infant with severe height retardation (-5.0 SD), typical growth hormone (GH)-deficient phenotype, and undetectable GH serum levels in response to three pharmacological stimuli. Diagnosis of isolated GH deficiency type 1A was confirmed by restriction endonuclease analysis of genomic DNA which pointed out GH-N gene deletion. The introduction of bio-methionyl-GH therapy in this patient was followed by a transient and clinically irrelevant appearance of low binding capacity GH antibodies as well as by a long-lasting catch-up growth (42.2 cm) which is continuing 44 months after beginning of treatment. This atypical pattern confirms that immune and growth response to exogenous GH in isolated GH deficiency 1A may be very heterogeneous.
Autoradiography↗
Recurrent nonsense mutations in the growth hormone receptor from patients with Laron dwarfism.
In addition to its classical effects on growth, growth hormone (GH) has been shown to have a number of other actions, all of which are initiated by an interaction with specific high affinity receptors present in a variety of tissues. Purification of a rabbit liver protein via its ability to bind GH has allowed the isolation of a cDNA encoding a putative human growth hormone receptor that belongs to a new class of transmembrane receptors. We have previously shown that this putative growth hormone receptor gene is genetically linked to Laron dwarfism, a rare autosomal recessive syndrome caused by target resistance to GH. Nevertheless, the inability to express the corresponding full-length coding sequence and the lack of a test for growth-promoting function have hampered a direct confirmation of its role in growth. We have now identified three nonsense mutations within this growth hormone receptor gene, lying at positions corresponding to the amino terminal extremity and causing a truncation of the molecule, thereby deleting a large portion of both the GH binding domain and the full transmembrane and intracellular domains. Three independent patients with Laron dwarfism born of consanguineous parents were homozygous for these defects. Two defects were identical and consisted of a CG to TG transition. Not only do these results confirm the growth-promoting activity of this receptor but they also suggest that CpG doublets may represent hot spots for mutations in the growth hormone receptor gene that are responsible for hereditary dwarfism.
Dwarfism↗
Exclusion of linkage between the collagenase gene and generalized recessive dystrophic epidermolysis bullosa phenotype.
Generalized recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited autosomal disease characterized by dermolytic blister formation. Enhanced collagenase and/or abnormal collagenase have been reported in skin from affected patients, suggesting that collagenase could be responsible for the absence of anchoring fibrils in this disorder. We used a genetic linkage approach to test the hypothesis that this disease is due to a defect in the collagenase gene in nine affected families. Analysis of amplified genomic DNA fragments of the collagenase gene by means of denaturing gradient gel electrophoresis (DGGE) allowed us to detect intragenic polymorphisms, which were subsequently characterized by direct genomic sequencing. Segregation analysis of these polymorphic sites showed exclusion of linkage between the collagenase gene and generalized RDEB phenotype in a family with consanguineous parents and three affected children. However, the possibility of linkage with the collagenase gene in the other eight families tested could not be excluded. The genetic markers described here provide a tool for investigating genetic linkage in other affected families. Overall, our results show that generalized RDEB can be caused by a defect in a gene other than the collagenase gene, and support the hypothesis that the genetic defect lies in abnormal anchoring fibril formation.
Base Sequence↗
Sickle cell anemia in the Tunisian population: haplotyping and HB F expression.
Thirty-three Tunisian patients, homozygous for Hb S, were examined. Haplotyping using nine restriction sites in the beta-globin gene cluster revealed that the most common type is the Benin type [---- + - + - +] which occurs at a frequency of 0.94% (31 cases); only one patient was homozygous for an atypical haplotype which shows some differences with the Benin haplotype at sites 1, 5, 6, and 8 [+ ---- + + + +]; the two remaining patients were assumed to be double heterozygotes for the Benin and atypical haplotypes. The presence of the atypical haplotype suggested a double origin of the beta S gene in Tunisia. Moreover, a heterogeneity in the Hb F production was observed, ranging between 2 to 16%, whereas the G gamma-globin expression was remarkably homogeneous in our patients with a normal amount approaching 40%. These results suggested the presence of a combination of several control factors.
Adolescent↗
Screening for non-delta F508 mutations in five exons of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in Italy.
Analysis of exons 10, 11, 14a, 15, and 20 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by denaturing-gradient-gel electrophoresis (DGGE) allowed the identification of mutations causing cystic fibrosis (CF) in 25 of 109 non-delta F508 chromosomes, as well as identification of a number of polymorphisms and sequence variations. Direct sequencing of the PCR fragments which showed an altered electrophoretic behavior not attributable to known mutations has led to the characterization of four new mutations, two in exon 11, and one each in exons 15 and 20. Screening for the different mutations thus far identified in our patients by the DGGE analysis and other independent methods should allow detection of about 70% of the molecular defects causing CF in Italy. Mutations located in exons 11 and 20 account for at least 30% of the non-delta F508 mutations present in Italian CF patients.
Base Sequence↗
[The cystic fibrosis gene: mutation and the function of CFTR protein].
The spectrum of mutations identified in cystic fibrosis patients includes a major defect (delta F508), found in 70% of CF chromosomes from French patients, and a large number of infrequent mutations. This significant heterogeneity of molecular defects precludes large scale screening for the disease at the time being. The presumptive structure of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR), the membrane protein encoded by the CF gene, is briefly recalled. The role this protein is thought to play in the regulation of chloride ion transport across epithelial membranes is discussed, along with the new insights into the molecular mechanisms of CF gained as a result of identification of the CF gene.
Cystic Fibrosis↗
Mutations in the p53 gene in myelodysplastic syndromes.
We have examined p53 alleles in 151 DNAs from patients with myelodysplastic syndrome using single-strand conformation polymorphism analysis of polymerase chain reaction products. We focused our study on the four highly conserved regions of the p53 gene and detected five patients with aberrantly migrating fragments. We confirmed the putative mutation in each case by direct sequencing analysis. Of these five patients, three had chromosome 17 monosomy associated with p53 mutation, one patient showed one mutated p53 allele and one wild-type allele, and the last patient demonstrated only the mutant allele, suggesting a homozygous state. Unlike many other types of human cancers, point mutations in the p53 tumor-suppressor gene appear to be a rare event in myelodysplastic syndromes.
Alleles↗
[Molecular genetic of growth hormone resistance syndrome].
The cloning of a putative growth hormone receptor (GH-R) cDNA has opened new approaches for the understanding of the molecular basis of GH insensitivity in humans. This molecule belongs to a new class of transmembrane receptors including prolactin, granulocyte-macrophage colony stimulating factor, erythropoeitin and some interleukin receptors. Although the domains responsible for signal transduction have not yet been identified, the molecular study of a GH-resistance syndrome described by Laron et al. should provide insight into the structure-function relationships of the GH-R and related receptors. This autosomal recessive disorder is characterized by very low serum levels of Insulin-Like Growth Factor I (IGF-I), despite increased secretion of GH with normal activity. Two approaches can be used to test the involvement of the GH-R in this syndrome. The first one, which is indirect, is performed through linkage analysis between GH-R and Laron phenotype; this allowed us to incriminate the GH-R gene in this syndrome. The second approach consists in the identification of molecular defects in the GH-R gene of patients with Laron syndrome; this allowed the detection of a partial gene deletion, two stop codons and one missense mutation. The short stature of the Pygmee population could be related to the Laron syndrome because individuals from this population are also resistant to GH therapy. Therefore, it seems interesting to search for molecular variations of the GH-R gene in this population. Nevertheless, preliminary results indicate that the GH-R gene is not directly involved in this particular short stature condition.(ABSTRACT TRUNCATED AT 250 WORDS)
Black People↗
A dimorphic 4-bp repeat in the cystic fibrosis gene is in absolute linkage disequilibrium with the delta F508 mutation: implications for prenatal diagnosis and mutation origin.
The gene causing cystic fibrosis (CF) has been recently cloned, and the major mutation (delta F508) accounting for approximately 70% of CF chromosomes has been uncovered. We have identified at the 3' end of intron 6 in the CF gene a 4-bp tandem repeat (GATT) that exhibits interesting features. First, PCR screening of 103 normal individuals revealed that the repeat exists only in two polymorphic allelic forms, either as a hexamer or a heptamer. These two alleles are in Hardy-Weinberg equilibrium and predict a heterozygote frequency of 41% (p[seven repeats] = .71; q [six repeats] = .29). Second, the allele with six repeats was found linked to delta F508 on all 76 CF chromosomes investigated, demonstrating strong linkage disequilibrium and suggesting that delta F508 had originated on the gene bearing six repeats. Third, when the repeat alleles are linked to the DNA markers XV2c and KM19, extended haplotypes are generated. These new haplotypes become informative in situations in which prenatal diagnosis cannot be performed solely with XV2c and KM19. Since this repeat marker is located in the CF gene and would be very less likely to recombine with the gene, it can serve as a valuable DNA marker for haplotype analysis. A possible crossover, however, was identified between XV2c and KM19, transferring delta F508 to a different haplotype.
Alleles↗
The molecular genetic analysis of hemophilia A: a directed search strategy for the detection of point mutations in the human factor VIII gene.
A directed-search strategy for point mutations in the factor VIII gene causing hemophilia A was used to screen eight potentially hypermutable CpG dinucleotides occurring at sites deemed to be of functional importance. Polymerase chain reaction-amplified DNA samples from 793 unrelated individuals with hemophilia A were screened by discriminant oligonucleotide hybridization. Point mutations were identified in 16 patients that were consistent with a model of 5-methylcytosine (5mC) deamination. Four new examples of recurrent mutation were demonstrated at the following codons: 336 (CGA----TGA), 372 (CGC----TGC), 372 (CGC----CAC), and 1689 (CGC----TGC). These are functionally important cleavage sites for either activated protein C or thrombin. Further novel C----T transitions were identified in the remaining arginine codons screened (-5, 427, 583, 795, and 1696), resulting in the creation of TGA termination codons. Differences in mutation frequency were found both within and between the CpG sites and between ethnic groups. These differences are assumed to be due to differences in the level of cytosine methylation at these sites, although direct evidence for this inference is lacking.
Base Sequence↗