[Unilateral partial deferential agenesia and CFTR gene composite heterozygoty (deltaF508/V938G)]. / Agénésie déférentielle partielle unilatérale et hétérozygotie composite du gène CFTR (DeltaF508/V938G).

This is a case report of a thirty-year-old-man consulting for a primary infertility that was diagnosed four years ago. Andrologic exam was normal. Two spermograms found normal spermatic parameters. An uro-genital echography with a RMI showed that a unilateral agenesia of the left vas deferens in the pelvic portion. Then, a composite heterozygoty of the CFTR gene (DeltaF508/V938G) was found. This is the first time that the association of these two mutations has been described. This case also makes it possible to wonder about the need for realizing, or not, a systematic basis imagery (ultrasound examination in first), in the event of infertility of the couple. In this context, the discovery of an echographic anomaly made it possible to identify CFTR mutations, whose physiopathological implication in the infertility can be discussed (CFTR related disorders)...

The covalent structure of dog myoglobin.

The primary structure of the myoglobin of the domestic dog (German shepherd) was studied. Tryptic and thermolytic peptides were compared with the sequence of other known myoglobins; the stepwise automatic Edman's degradation of the whole globin and also the chymotryptic digestion of the median fragment obtained by CNBr cleavage completed this sequence. Comparison of the established dog myoglobin structure with those from other carnivora shows 16 differences versus badger, 20 versus harbour seal and 15 versus California sea lion.

Inactivation of unbound rat liver glucocorticoid receptor by N-alkylmaleimides at sub-zero temperatures.

A series of N-alkylmaleimides was shown to inactivate effectively the rat liver glucocorticoid receptor at neutral pH. A partial purification of the unbound cytosolic receptor by protamine sulfate precipitation and a careful stabilization of the essential thiol by dithiothreitol and sodium molybdate before the alkylation step appeared essential to obtain pseudo-first-order kinetics. Moreover, performing the experiment at -12 degrees C in buffer containing 40% glycerol as antifreeze agent resulted in increased receptor stabilization and a slowing-down of the inactivation process, which could then be more accurately studied. This process was demonstrated to be dose- and pH-dependent in the case of N-ethyl- and N-nonylmaleimides. Furthermore, comparison of the various N-alkylmaleimides revealed a striking increase of receptor inactivation with increasing chain length of the maleimide derivative. Full protection against inactivation was afforded by previous [3H]dexamethasone binding on the receptor. Long-chain N-alkylmaleimides inactivated by beta-mercaptoethanol were still able to inhibit the [3H]-dexamethasone binding noncovalently. Likewise N-nonylsuccinimide was shown to compete with [3H]dexamethasone for receptor binding. It is suggested that the chain length effect observed in the inactivation process is related to nonpolar interactions in the binding of maleimides to the receptor prior to the irreversible alkylation of sulfhydryl groups. These groups lie in a hydrophobic environment, probably in the steroid binding site itself.

Identification of novel VMD2 gene mutations in patients with best vitelliform macular dystrophy.

ABSTRACT We report five novel VMD2 mutations in Best's macular dystrophy patients (S16F, I73N, R92H, V235L, and N296S). An SSCP analysis of the VMD2 11 exons revealed electrophoretic mobility shifts exclusively in exons 2, 3, 4, 6 and 8. Direct sequencing indicated that these shifts are caused by mono-allelic transition in exons 2, 4, 6, 8 and transversion in exons 3 and 6. Five novel "silent" polymorphisms are also reported: 213T>C, 323C>A, 1514A>G, 1661C>T, and 1712T>C. Hum Mutat 17:235, 2001.

[Specificity of thermolysin action on dog myoglobin]. / Spécificité d'action de la thermolysine sur la myoglobine de chien

The proteolytic specificity of thermolysin has been studied by quantitative analysis of an enzymic digest of dog myoglobin. Results confirm main specificities of thermolysin towards Phenylalanine, Isoleucine, Leucine or Tyrosine bonds; the influence of neighbourhood was also determined and the conclusions are in a good agreement with the known structure of the active site of thermolysin.

Physico-chemical properties and evidence for electrophoretic variants of rat transcortin.

Isolation of rat plasma transcortin was carried out by affinity chromatography, as previously described for human. The protein was shown to be pure by PAGE and one single N-terminal amino acid was identified (Ser), which suggested that the protein molecule has a single polypeptide chain. This assumption is supported by SDS-PAGE. The amino acid composition was reported and compared with the one of human transcortin. The purified protein always migrated in PAGE (with or without SDS) as a double band; the faster component being more intense than the slower one. Whether transcortin was free or bound to corticosterone, the same aspect was observed. Molecular weight of these two variants were determined by SDS-PAGE as 65,900 and 75,800. Polymers only appeared after irreversible denaturation of the protein, as previously described for human transcortin. Various other physical parameters were determined: a sedimentation coefficient of 3.71 S +/- 0.18 was calculated by ultracentrifugation in sucrose gradient, association constants at 4 degrees C for corticosterone and cortisol (2.7 X 10(9) M-1 and 4.2 X 10(8) M-1, respectively).

Use of denaturing HPLC and automated sequencing to screen the VMD2 gene for mutations associated with Best's vitelliform macular dystrophy.

We identified three novel VMD2 mutations in patients with Best's macular dystrophy. DHPLC analysis of the 11 VMD2 exons revealed abnormal profiles in exon 8. Direct sequencing showed that these abnormal profiles were due to monoallelic transitions and transversions. We also found three polymorphic sequence changes that have been reported previously and annotated to an online database (http://www.uni-wuerzburg.de/humangenetics/vmd2.html).

Altered rep-1 expression due to substitution at position +3 of the IVS13 splice-donor site of the choroideremia (CHM) gene.

PURPOSE: To characterize the effect on mRNA splicing of a yet undescribed mutation located in intron 13 splice-donor sequence (IVS13 + 3A --> C) in the Rab-Escort-protein 1 gene of a patient with choroideremia. METHODS: The base substitution was firstly detected by the Single Strand conformation analysis from genomic DNA. A REP-1 cDNA region encompassing exons 10-14 was then specifically amplified from lymphocytes-derived mRNA. RESULTS: We could demonstrate that this substitution affects REP-1 RNA processing. The patient revealed only one aberrantly spliced mRNA lacking exon 13 and no normal transcript. CONCLUSION: The skipping of exon 13 results in the creation of a stop codon at the misspliced junction. This is the first case of nucleotide substitution at the +3 position of a splice donor site so far described in choroideremia.

Genotypes of cytochrome P450 and clinical response to clomipramine in patients with major depression.

The genetic cytochrome P450 polymorphism is reported in factors affecting the individual response to drugs. The interindividual variation at steady-state levels or also in elimination of drugs, finds an explanation in genetic differences in the metabolism. In particular, activities of the P450-IID6 isoenzyme are related to the sparteine/debrisoquine oxidation polymorphism. Phenotyping such a system has been proposed to analyse variability in the tricyclic antidepressant level. To analyse clinical relevance of a pharmacogenetic approach, we studied the cytochrome P450 CYP2D6 genotypes and the clinical responses to clomipramine in 21 hospitalised patients who met DSM-III-R criteria for major depression. Three patients were predicted as poor metabolizers. We suggested a limitation of clomipramine (CMI) hydroxylation in poor metaboliser (PM) patients which is balanced by a desmethylation. The clinical efficacy pattern does not differ in poor metaboliser and early metaboliser patients. Firstly, there is no significant differences in the evolution of scores on MADRS and specific retardation scale into the two groups. Secondly, outcome of side effects does not occur more frequently in PM patients. Clinical relevance of such an approach needs further study.

Length variations of the poly(T) tract at the exon 3 splice acceptor site of the choroideremia gene.

By using the single strand conformational analysis to search for point mutations in the choroideremia gene, we have identified an intronic polymorphism within the intron 2 of the CHM gene. We have studied the frequency of this polymorphism in the population from South of France.

[Pendred's syndrome. Current features]. / Le syndrome de Pendred. Aspects actuels.

Introduction Pendred's syndrome is a recessive autosomal disease, traditionally defined as the association of deaf-mutism, goiter and dysfunctional iodide organization revealed by the perchlorate discharge test. It represents 4 to 10% of the causes of congenital hypoacusis. Although described more than a 100 years ago, the association of thyroid and cochleo-vestibular damage remained unclear for many years. Genetic abnormalities Progress in molecular biology has revealed that the disease is related to alterations in the PDS gene situated on chromosome 7. The PDS gene is responsible for the production of pendrine, protein involved in anion (l-, Cl-) transportation, notably in the apical pole of the thyreocyte and the cochlear duct, where the endolympha is produced. Practical implications The truncation of pendrine related to the genetic alterations be responsible for the morpho-functional alterations in the cochlear apparatus and the thyroid. In this perspective, Pendred's syndrome would appear as a genetic disorder in anion transportation.

[Cutaneous manifestations of zinc deficiency in ethylic cirrhosis]. / Manifestations cutanées du déficit en zinc dans la cirrhose éthylique.

Thirty-three patients with alcoholic cirrhosis (AC), selected on widely recognized criteria (16, 57), were investigated prospectively for cutaneous manifestations of zinc deficiency. The patients were divided into 3 groups: group A (n = 12): AC without skin lesions; group B (n = 12): AC with skin lesions responsive to a zinc-free topical treatment or resistant to enteral zinc sulfate intake; group C (n = 9): AC with skin lesions cured by oral zinc replacement therapy alone. The lesions observed in group C were studied microscopically. Data concerning zinc metabolism (Zn concentrations in plasma, red cells, urine and hair; alkaline phosphatase values), biochemical criteria of AC (plasma serum-albumin concentration, IgA/transferrin ratio) and a malabsorption test (xylosemia 120 min after oral absorption of D-xylose 25 g) were compared by the variance analysis method. A control group (D, n = 12) was used as reference. Few cases of cutaneous manifestations of zinc deficiency in AC patients have been published. In more than one half of the 15 or so we found in the literature, an aggravating factor (total parenteral nutrition, digestive tract surgery) had to be taken into account. In this prospective study 9 new cases in which AC was the only cause of zinc deficiency are reported. A clinical picture similar to acrodermatitis enteropathica with peribuccal bullous lesions was observed in only one patient. In all other cases the patients presented with a cracked and reticulated eczema on the extensor aspect of the limbs and (often erosive) in the perianal and genital regions. The eczema was associated with cheilitis, glossitis, stomatitis, alopecia and, seldom, ungual Beau's lines. Disorders of behaviour, diarrhoea and bouts of lever regressing under zinc replacement therapy were frequent. Histology was not very specific, except for the presence of necrotic areas in the stratum germinativum, sometimes associated with small subcorneal pustules containing altered polymorphonuclears. In every case, it was the rapid regression of symptoms under zinc sulfate treatment that confirmed the diagnosis. Plasma zinc concentrations were most significantly decreased in all AC groups as compared to controls (61.2 +/- 19.4 vs 97.8 +/- 10.4 micrograms/100 ml) and also in AC patients with skin manifestations of zinc deficiency as compared to the other AC patients (44.4 +/- 9.2 vs 66.5 +/- 18.8 micrograms/100 ml) table V). Changes in serum-albumin levels and in hepatocellular function were parallel to changes in plasma zinc concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)

[Correlation of genitourinary abnormalities, spermiogram and CFTR genotype in patients with bilateral agenesis of the vas deferens]. / Corrélation entre anomalies génito-urinaires, spermogramme et génotype CFTR chez les patients atteints d'une agénésie bilatérale des canaux déférents.

OBJECTIVES: To evaluate the frequency of urogenital ultrasound and spermatic abnormalities in patients with bilateral vas deferens agenesis according to the presence or absence of CFTR gene mutation. METHODS: In 41 patients with bilateral vas deferens agenesis confirmed by surgical exploration between 1988 and 1997, renal and seminal vesicle anomalies were investigated by ultrasonography. Spermatic parameters (pH, fructose and ejaculate volume) were also studied, together with sweat chloride assay and PCR of mutations on exons 3, 4, 7, 9, 10, 11, 13, 14b, 17b, 19, 20 and 21 of the CFTR gene. RESULTS: None of the 8 patients with a renal anomaly presented a CFTR gene mutation, versus 23 out of 33 patients without a renal anomaly (p < 0.02). Seminal vesical anomalies were not more frequent in patients with or without mutations (11/20 versus 13/19, p = NS), except for composite heterozygous patients (with 2 mutations: 8/13 versus 4/11, p = NS). Spermatic parameters (pH < 7.2, fructose < 1 g/l and volume < 2 ml) could not distinguish between patients with or without renal or seminal vesical anomalies or mutation, except for patients with pH < 7.2, who presented fewer renal anomalies (2/25 versus 6/16, p < 0.05) and a higher incidence of gene mutation (19/25 versus 5/12, p < 0.01). CONCLUSION: Renal agenesis is considered to be pathognomonic of a developmental anomaly. Unlike a seminal vesical anomaly, a semen volume < 2 ml or fructose < 1 g/l, pH less than 7.2 is a nonspecific parameter, but more frequently present in patients with CFTR mutation.

Inhibition of glucocorticoid receptor transformation, subunit dissociation, and temperature-dependent inactivation by various N-substituted maleimides.

A series of N-substituted maleimides were synthesized, and their effect on the activation to the DNA binding state of the rat liver glucocorticoid receptor was studied. Unactivated (preincubated at 0 degrees C) cytosolic [3H]triamcinolone acetonide-receptor complexes were pretreated with various N-alkylmaleimides at 0 degrees C and then heated at 25 degrees C and assayed for DNA-cellulose binding. No inhibition of the DNA binding activity was observed with either N-ethylmaleimide or N-substituted maleimides bearing an ionizable substituent, like N-(omega-carboxyalkyl)maleimides and N-[2-(trimethylammonio) ethyl]maleimide. On the contrary, treatment with long-chain alkylmaleimides like N-heptylmaleimide resulted in significant inhibition. The highest inhibition was obtained with N-benzylmaleimide and, to a lesser extent, N-(ethylphenyl)-maleimide, whereas N-benzylsuccinimide was ineffective. Treatment of cytosol containing unactivated glucocorticoid complexes at 3 degrees C with N-benzymaleimide also prevents the temperature-mediated conversion of 8S receptor to 4S. Moreover, N-benzylmaleimide was able to inhibit the inactivation of the receptor steroid-binding activity caused by heat. N-Benzylmaleimide shares with molybdate ions the ability to inhibit glucocorticoid receptor activation, dissociation, and inactivation. However, their respective mechanisms of action are probably distinct, since their effects on receptor inactivation appear additive. It is suggested from the comparison of the various maleimides tested that the sulfhydryl groups essential for receptor activation and dissociation lie in a rather nonpolar environment including aromatic amino acid(s).

Covalent chromatography by thiol-disulfide interchange of the highly-purified non-transformed rat liver glucocorticoid-receptor.

Highly-purified non-transformed rat liver [3H]triamcinolone acetonide-receptor complex was shown to be covalently adsorbed on activated thiol sepharose 4B, a reactive sulfhydryl matrice. Elution by mercaptoethanol in excess and inhibition of binding by previous treatment of the complex with N-ethylmaleimide clearly demonstrated the specificity of the binding by thiol disulfide interchange. The transformed [3H]triamcinolone acetonide-receptor complex, partially purified by DNA-cellulose chromatography, was also retained on activated thiol sepharose 4B. The physicochemical characteristics of both the transformed and non-transformed glucocorticoid receptor complexes eluted from the covalent chromatography column were studied by HP size exclusion chromatography on a TSK G 3000 SW column and were found to be identical to those of the starting complexes. These results provide direct evidence for accessible sulfhydryl groups on the glucocorticoid receptor complex surface, probably distinct from the steroid binding essential sulfhydryl group.

Congenital bilateral absence of the vas deferens (CBAVD) and cystic fibrosis transmembrane regulator (CFTR): correlation between genotype and phenotype.

To assess better the link between congenital bilateral absence of the vas deferens (CBAVD) and cystic fibrosis (CF), we compared sweat chloride values, analysis of the CFTR intron 8 poly(T) tract length and analysis of 10 exons in a population of 38 patients with CBAVD. The data indicate that this population can be divided into three groups of patients. In the first group of 15 patients with abnormal sweat chloride (> 60 mmol/l), the frequency of CF mutations is high. In the second group of 18 patients with equivocal sweat chloride (between 40 and 60 mmol/l), the frequency of the 5T variant is high; 6 patients have a delta F508 mutation and a 5T variant and 1 patient is homozygous for the 5T variant; a 5T variant has been detected in 3 other patients, and a delta F508 mutation in another patient. A third group of 5 patients is probably not related to CF: these patients have other congenital abnormalities of the urogenital tract, low chloride values (< 40 mmol/l) and apparently no abnormality of the CF gene.