Novel SDHD germ-line mutations in pheochromocytoma patients.

BACKGROUND: SDHD germ-line mutations predispose to pheochromocytoma (PCC) and paraganglioma (PGL). MATERIAL AND METHODS: The incidence and types of SDHD germ-line mutations are determined in 70 patients with apparently sporadic adrenal and extra-adrenal PCC. RESULTS: SDHD sequence variants were identified in the germ line of five patients. Two of three novel mutations were in exon 1 and one in exon 3. One patient had a codon 1 missense mutation (M1K) and a concurrent 3-bp deletion in intron 1. Three of 10 family members had only the exon 1 mutation, whereas one had only the intron 1 mutation. The other exon 1 mutation resulted from a deletion of nucleotides 28-33 with a 12-bp in-frame insertion (c.28_33 del ins TAGGAGGCCCTA). This mutation generated a premature stop codon after codon 9 and was also present in the brother who had a bilateral PCC. The third patient with a carotid body tumour, with an abdominal and a thoracic PGL had a 12-bp deletion in exon 3 (codons 91-94, c.271_282 del). Her father carried the same mutation and had bilateral carotid body tumours. Two further patients, one with six PGL, carried a previously described H50R polymorphism, whose disease-specific relevance is currently unclear. The three patients with bona fide SDHD mutations were younger than those without germ-line mutations. CONCLUSION: SDHD germ-line mutations are rare in patients with PCC, but their identification is an important prerequisite for the clinical care and appropriate management of affected individuals and their families.

Cytotoxic activity of camptothecin and paclitaxel in newly established continuous human medullary thyroid carcinoma cell lines.

At the time of diagnosis, more than one quarter of patients with medullary thyroid carcinoma (MTC) has distant metastases. Only few of these patients can be cured by surgery. Standard chemotherapy is characterized by low response rates and short response time. The establishment of eight human MTC cell lines provides a new basis for in vitro investigation of cytotoxic drugs. Camptothecin (CPT) and paclitaxel, which never have been investigated in the treatment of MTC, were tested for their cytotoxic profile in comparison with the clinically ineffective dacarbazine. Eight MTC cell lines were established from seven patients with MTC. IC(50) values were calculated from dose-response relationships using cell counts and a formazan dye assay (WST-1). IC(50) values were 3.5 +/- 1.2 nmol/liter for CPT and 8.2 +/- 1.9 nmol/liter for paclitaxel. Dacarbazine showed no reduction of cell proliferation at concentrations 10-fold higher than clinically achievable. Given peak plasma concentrations of 65 +/- 20 nmol/liter for CPT and 1 micro mol/liter for paclitaxel, these promising in vitro results provide a basis for the performance of clinical trials in patients with advanced MTC.

Sporadic versus familial medullary thyroid microcarcinoma: a histopathologic study of 50 consecutive patients.

By means of calcitonin screening programs, sporadic and hereditary medullary thyroid carcinoma (MTC) can be detected at an early stage. We investigated the histopathologic findings of 16 familial (mean age 32 +/- 21 years, female/male ratio 1.6:1) and 34 sporadic (mean age 58 +/- 15 years; female/male ratio 2.4:1) MTCs with stage T1 comparatively. Patients with hereditary tumors were younger. Hereditary tumors were more often found multifocal (13 of 16 vs 3 of 34; p < 0.001), bilateral (11 of 16 vs 3 of 34; p < 0.001), displaying desmoplastic stroma (14 of 16 vs 19 of 34; p = 0.02), and accompanied by C cell hyperplasia (16 of 16 vs 24 of 34; p = 0.01), but all of these factors were present in some sporadic patients. Only tumors with desmoplastic stroma showed lymph node metastasis, which was observed in eight of the 50 patients. After surgery all patients showed permanent normalization of calcitonin levels. We conclude that 1) morphologic parameters considered to indicate familial MTC risk are of no value in the individual patient, 2) many sporadic MTCs develop on the background of CCH, 3) tumors with desmoplastic stroma are more likely to develop lymph node metastasis, and 4) early detection of MTC permits curative surgery in the majority of patients.

Evaluation of the fragile X (FRAXA) syndrome with methylation-sensitive PCR.

The fragile X (FRAXA) syndrome is the most common form of inherited mental retardation in males. Its peculiar pattern of inheritance results from the parent of origin-specific expansion of a CGG-repeat within the FMR1 gene on the X chromosome. In patients, gene function is abolished by hypermethylation of the promoter and the massively expanded repeat. We have developed a methylation-sensitive polymerase chain reaction (MS-PCR) strategy that combines repeat-length and methylation analysis of the CGG-repeat and the promoters of the FMR1 and XIST genes. The allelic methylation of the latter opposes that of the FMR promoter and serves as an internal control and standard for semiquantitative analyses. This system enables the delineation of 11 distinct patterns encountered in nonaffected, carrier, and affected males and females. We have evaluated our system on well-defined samples with different FMR1 mutations and have used it for the diagnostic evaluation of 253 male and 80 female probands. In the male group, we have identified five full mutations, and three gray-zone and premutation alleles with 54, 55, and 62 repeats, respectively. The female group consists of 33 normal homozygote and 41 heterozygote individuals, two of whom harbor a gray-zone allele with 47 repeats, none with a premutation, and six with a full mutation. Our MS-PCR approach allows the currently most comprehensive diagnostic evaluation of the FRAXA syndrome in a cost- and time-efficient fashion. In addition, it is a valuable tool for the analysis of clonality and skewing phenomena in females.

Methylation changes in promoter and enhancer regions of the WT1 gene in Wilms' tumours.

Although the WT1 gene has been implicated in the aetiology of Wilms' tumour, mutations in WT1 are found only in minority of the tumours. DNA methylation of regulatory elements represents another possibility of modulation of gene expression. We studied methylation in the promoter and enhancer regions of the WT1 gene in 34 Wilms' tumour patients by the polymerase chain reaction on HpaII-digested DNA and by the bisulphite method. No methylation was detected in the promoter region in either tumour or normal kidney or blood DNA samples. In contrast, a HpaII site in the enhancer region was at least partially methylated in normal kidney and blood DNA samples and in about one-third of the tumours, while the majority of tumours showed no methylation. The differential methylation in the enhancer region of the WT1 gene may indicate that methylation of this element can play a role in the regulation of this gene.

C-cell hyperplasia and medullary thyroid carcinoma in patients routinely screened for serum calcitonin.

Routine screening of calcitonin serum levels in patients with nodular thyroid disorders has led to an increased rate of total thyroidectomies. We investigated prevalence and interrelationship of C-cell hyperplasia (CCH) and medullary thyroid carcinoma (MTC) in patients with thyroid and parathyroid disorders that showed increased calcitonin serum levels detected by routine screening. Within two years, 30 (mean age, 60 +/- 14 years) of 667 patients had a pentagastrin-stimulated calcitonin level of more than 100 pg/mL. All 30 underwent total thyroidectomy and were tested for germ-line mutations of the ret protooncogene. Entire surgical specimens were blocked, and C-cell disorders were assessed using conventional histology and immunohistochemistry. C-cell hyperplasia was defined by the presence of more than 50 C cells/l low-power field in both lobes and was classified as focal, diffuse, nodular, or neoplastic. Nineteen patients (female/male = 14/5) had MTC, and 11 males but no females had CCH only. Six of 16 patients with sporadic MTC had concomitant CCH. Three patients were index cases of new MTC families. We conclude that MTC with concomitant CCH is an unreliable marker for hereditary MTC risk and that CCH has a preneoplastic potential in the absence of germ-line mutations. In this series, CCH alone was not found in females.

alpha-1,4-D-glucan phosphorylase of gram-positive Corynebacterium callunae: isolation, biochemical properties and molecular shape of the enzyme from solution X-ray scattering.

The alpha-1,4-D-glucan phosphorylase from gram-positive Corynebacterium callunae has been isolated and characterized. The enzyme is inducible approx. 2-fold by maltose, but remarkably not repressed by D-glucose. The phosphorylase is a homodimer with a stoichiometric content of the cofactor pyridoxal 5'-phosphate per 88-kDa protein subunit. The specificity constants (kcat/Km, glucan) in the directions of glucan synthesis and degradation are used for the classification of the enzyme as the first bacterial starch phosphorylase. A preference for large over small substrates is determined by variations in the apparent binding constants rather than catalytic-centre activities. The contribution of substrate chain length to binding energy is explained assuming two glucan binding sites in C. callunae phosphorylase: an oligosaccharide binding site composed of five subsites and a high-affinity polysaccharide site separated from the active site. A structural model of the molecular shape of the phosphorylase was obtained from small-angle solution X-ray scattering measurements. A flat, slightly elongated, ellipsoidal model with the three axes related to each other as 1:(0.87-0.95):0.43 showed scattering equivalence with the enzyme molecule. The model of C. callunae phosphorylase differs from the structurally well-characterized rabbit-muscle phosphorylase in size and axial dimensions.

Reaction engineering aspects of alpha-l,4-D-glucan phosphorylase catalysis : comparison of plant and bacterial enzymes for the continuous synthesis of D-glucose-1-phosphate.

Some important process properties of alpha-l,4-D-glucan phosphorylases isolated from the bacterium Corynebacterium callunae and potato tubers (Solatium tuberosum) were compared. Apart from minor differences in their stability and specificity (represented by the maximum degree of maltodextrin conversion) and a 10-fold higher affinity of the plant phosphorylase for maltodextrin (K (M) of 1.3 g/L at 300 mM of orthophosphate), the performances of both enzymes in a continuous ultrafiltration membrane reactor were almost identical. Product synthesis was carried out over a time course of 300-400 h in the presence or absence of auxiliary pullulanase (increasing the accessibility of the glucan substrate for phosphorolytic attack up to 15-20%). The effect of varied dilution rate and reaction temperature on the resulting productivities was quantitated, and a maximum operational temperature of 40 degrees C was identified.

Efficient downstream processing of maltodextrin phosphorylase from Escherichia coli and stabilization of the enzyme by immobilization onto hydroxyapatite.

Downstream processing by biospecific chromatography of maltodextrin phosphorylase from Escherichia coli, overexpressed in E. coli, was substantially improved by a novel approach using ceramic hydroxyapatite. Wild-type and a less active mutant enzyme were purified from crude bacterial cell extracts in one efficient separation step that yielded phosphorylase in purity > 95% in at least 90% recoveries. At pH 6.9 and 25 degrees C, wild-type and mutant phosphorylases eluted from the hydroxyapatite column at a phosphate concentration of 0.4 M whereas calcium ions failed to displace the enzymes. The dynamic capacity for phosphorylase binding in the presence of bulk proteins was approximately 3 mg enzyme ml-1 matrix. The interaction of E. coli phosphorylase with hydroxyapatite seems to be mediated by surface amino groups, so that the bound enzyme retained almost full catalytic activity. Compared to the soluble enzyme, immobilization onto hydroxyapatite resulted in a more than 30-fold stabilization of wild-type phosphorylase against thermal and proteolytic inactivation and thus could improve the operational stability of phosphorylase during conversion of polysaccharide to glucose 1-phosphate.