Biomedical subjects

Beat Steinmann

Publications and source records attributed to Beat Steinmann.

31 records · Page 2Linked to original sources

PubMed2003

Two-dimensional NMR spectroscopy of urinary glycosaminoglycans from patients with different mucopolysaccharidoses.

Patients with different types of mucopolysaccharidoses (MPS) lack specific lysosomal enzymes, which leads to tissue accumulation and urinary excretion of glycosaminoglycans (GAGs). Since little is known about the molecular composition of the excreted GAG fragments, we used two-dimensional [1H,13C]-correlation nuclear magnetic resonance (NMR) spectroscopy for a detailed analysis of the urinary GAGs of patients with MPS types I, II, IIIA, IVA and VI. The method revealed that the molecular structures of the excreted GAGs, i.e. heparan sulfate (HS), dermatan sulfate (DS), chondroitin sulfate (CS), and keratan sulfate (KS) are clearly distinct for the different MPS types. The chain terminal residues that are the normal substrates for the defective enzymes constitute characteristic sets of signals for each MPS type. The GAG chains show variations in carbohydrate composition and sulfation patterns that can be related to the different MPS types and clinical features. For example, two patients with MPS IIIA (M. Sanfilippo) with signs of CNS degeneration but only mild somatic features excrete a highly sulfated variant of HS, resembling HS in porcine brain, whereas a patient with MPS I (M. Scheie) and two patients with MPS II (M. Hunter), who present primarily with coarse facial features, joint contractures and skeletal deformities excrete a different type of HS with lower sulfation. In another case study, a patient with MPS IVA (M. Morquio), who presented mainly with skeletal dysplasia, excreted not only excessive amounts of KS but also a highly sulfated CS variant, resembling CS in articular cartilage. The high-resolution NMR analysis of urinary GAGs presented here for the first time provides a solid basis for future studies with a larger number of patients to further explore pathogenesis and course of the MPS diseases.

Adult↗

PubMed2003

Intestinal glucose transport: evidence for a membrane traffic-based pathway in humans.

BACKGROUND & AIMS: The presence of glucose transporter 2 (GLUT2) molecules in the basolateral membrane of enterocytes has long been considered to be of major importance for intestinal glucose absorption. The aim of this study was to reevaluate the role of GLUT2 in a patient with congenital GLUT2 deficiency (Fanconi-Bickel syndrome, FBS). METHODS: Oral mono- and disaccharide tolerance tests including gaschromatographic determination of breath hydrogen concentrations were performed in an FBS patient. For comparison, a patient with a microsomal carbohydrate transport defect, glucose-6-phosphate translocase 1 (G6PT1) deficiency, and a control individual were investigated. RESULTS: No increase in breath hydrogen concentration was found in the GLUT2-deficient patient after a glucose load. In G6PT1 deficiency, basal hydrogen concentrations were repeatedly found to be elevated. CONCLUSIONS: From the fact that a GLUT2-deficient patient does not show any impairment of intestinal monosaccharide transport measurable by the hydrogen breath test, we conclude that mechanisms other than facilitative glucose transport by GLUT2 must be involved in the transport of monosaccharides at the basolateral membrane of enterocytes. When relating this observation to the high intestinal expression of human hexokinase, G6PT1, and glucose-6-phosphatase and to our results of oral carbohydrate tolerance tests in a G6PT1-deficient patient, there is evidence that a microsomal membrane traffic-based transport pathway, as recently suggested for GLUT2-deficient animals, also plays a major role in transcellular monosaccharide transport of the human intestine.

Adult↗

PubMed2003

Severe lymphatic microangiopathy in Fabry disease.

BACKGROUND: Lymphedema has been described in a few cases of Fabry disease. The etiology of lymphedema in Fabry disease is unknown. The aim of the study was to evaluate morphology and function of lymphatic microvessels in this disease. METHODS AND RESULTS: In five male patients with Fabry disease, the initial lymphatic microvessels of the skin were studied in vivo, using a nearly atraumatic technique of fluorescence microlymphography and measurement of lymph capillary pressure. In addition, five female patients heterozygous for Fabry disease and 12 healthy controls were studied. The maximum spread of the fluorescent macromolecular dye into the network of superficial skin lymphatics was increased in the three male patients presenting with lymphedema (25, 26, and 45 mm, respectively). In the two male patients without swollen legs, the maximum spread of the dye was 3 and 7 mm, respectively, and in the female patients 8.8 mm (range, 4-17 mm), whereas in the healthy controls it reached only 4.3 mm (range, 1-7 mm). Fragmentation of the microlymphatic network was found in all patients, but not in controls. In controls, the diameter of the microvessels varied in a very narrow range (45-75 microm); in patients, the range was 15-150 microm. In patients with lymphedema, microlymphatic hypertension was present. CONCLUSION: In patients with Fabry disease severe structural and functional changes of the initial lymphatics of the skin are present.

Adolescent↗

PubMed2003

Mass spectrometric analysis of human transferrin in different body fluids.

In this study, we present a versatile new procedure for the analysis of transferrin and its isoforms isolated from human body fluids such as serum, plasma, and cerebrospinal fluid. This method is based on a three-step procedure: (i) isolation of transferrins using anion-exchange chromatography with UV detection; (ii) concentration of the transferrin fraction; (iii) detection of the transferrins with liquid chromatography-electrospray mass spectrometry. Pre-analytical sample procedures can be omitted and no immunoaffinity columns or transferrin-specific immunoassays were used. Anticoagulants such as heparin, EDTA, citrate, and oxalate do not interfere with our analysis. According to their respective molecular masses, up to ten different isoforms of transferrin could be identified in a serum sample from a patient with a congenital disorder of glycosylation type Ia (CDG-Ia). The method was successfully applied to different pathological samples from patients with CDG-Ia, CDG-Ib, CDG-Ic, CDG-Ie, CDG-If, and CDG-IIa. Additionally, samples from alcohol consumers that were found with turbidimetric immunoassay to contain increased levels of carbohydrate-deficient transferrin were analyzed.

Body Fluids↗

PubMed2002

Quantification of single nucleotide polymorphisms: a novel method that combines primer extension assay and capillary electrophoresis.

We present a novel method for accurate quantification of single nucleotide polymorphism (SNP) variants in transcripts and pooled DNAs in a one-tube reaction. Our approach is based on single- nucleotide primer extension (SNuPE) and laser-induced fluorescence capillary electrophoresis (LIF-CE), and takes advantage of distinct mobilities of SNuPE products with different nucleotides incorporated at their 3' ends. The method, called SNuPE-ONCE, was tested on two polymorphisms and five mutations that comprised the three most frequent ( approximately 70%) nucleotide changes in the human genome (C/T, A/G, and A/T). The usefulness of the method was demonstrated by analyzing nonsense-mediated mRNA instability in fibroblasts. Our data show 1) that the method provides highly reproducible relative allele frequencies (SD<0.017) with a good accuracy (e.g. for heterozygotes 0.500 +/- 0.036, P = 0.01), depending on the sequence and the proportion of the SNP variants in the sample, and 2) that relative allele frequencies as low as 1% can be detected quantitatively and unambiguously. Our assay relies on a CE instrument available in many laboratories and offers a useful method for quantitative SNP genotyping as well as for a variety of expression studies.

Alleles↗

PubMed2002

Ten novel FBN2 mutations in congenital contractural arachnodactyly: delineation of the molecular pathogenesis and clinical phenotype.

Congenital contractural arachnodactyly (CCA) is an autosomal dominant condition that shares skeletal features with Marfan syndrome (MFS), but does not have the ocular and cardiovascular complications that characterize MFS. CCA and MFS result from mutations in highly similar genes, FBN2 and FBN1, respectively. All the identified CCA mutations in FBN2 cluster in a limited region similar to where severe MFS mutations cluster in FBN1, specifically between exons 23 and 34. We screened exons 22 through 36 of FBN2 for mutations in 13 patients with classic CCA by single stranded conformational polymorphism analysis (SSCP) and then by direct sequencing. We successfully identified 10 novel mutations in this critical region of FBN2 in these patients, indicating a mutation detection rate of 75% in this limited region. Interestingly, none of these identified FBN2 mutations alter amino acids in the calcium binding consensus sequence in the EGF-like domains, whereas many of the FBN1 mutations alter the consensus sequence. Furthermore, analysis of the clinical data of the CCA patients with characterized FBN2 mutation indicate that CCA patients have aortic root dilatation and the vast majority lack evidence of congenital heart disease. These studies have implications for our understanding of the molecular basis of CCA, along with the diagnosis and genetic counseling of CCA patients.

Adolescent↗

PubMed2002

Evaluation and application of denaturing HPLC for mutation detection in Marfan syndrome: Identification of 20 novel mutations and two novel polymorphisms in the FBN1 gene.

Mutations in the human fibrillin 1 gene (FBN1) cause the Marfan syndrome (MFS), an autosomal dominant connective tissue disorder. Knowledge about FBN1 mutations is important for early diagnosis, management, and genetic counseling. However, mutation detection in FBN1 is a challenge because the gene is very large in size ( approximately 200 kb) and the approximately 350 mutations detected so far are scattered over 65 exons. Conventional methods for large-scale detection of mutations are expensive, technically demanding, or time consuming. Recently, a high-capacity low-cost mutation detection method was introduced based on denaturing high-performance liquid chromatography (DHPLC). To assess the sensitivity and specificity of this method, we blindly screened 64 DNA samples of known FBN1 genotype exon-by-exon using exon-specific DHPLC conditions. Analysis of 682 PCR amplicons correctly identified 62 out of 64 known sequence variants. In three MFS patients of unknown FBN1 genotype, we detected two mutations and eight polymorphisms. Overall, 20 mutations and two polymorphisms are described here for the first time. Our results demonstrate 1) that DHPLC is a highly sensitive (89-99%, P = 0.05) method for FBN1 mutation detection; but 2) that chromatograms with moderate and weak pattern abnormalities also show false positive signals (in all 45-59%, P = 0.05); 3) that the difference in the chromatograms of heterozygous and homozygous amplicons is mostly independent of the type of sequence change; and 4) that DHPLC column conditions, additional base changes, and the amounts of injected PCR products influence significantly the shape of chromatograms. A strategy for FBN1 mutation screening is discussed.

Chromatography, High Pressure Liquid↗

PubMed2002

Analysis of the CTNS gene in patients of German and Swiss origin with nephropathic cystinosis.

The autosomal recessive lysosomal storage disorder, nephropathic cystinosis is characterized by impaired transport of free cystine out of lysosomes. The gene responsible for cystinosis, CTNS, consists of 12 exons and encodes a 55 kDa putative lysosomal membrane protein, called cystinosin. Up to now more than 55 different CTNS mutations have been described in cystinosis. We have analyzed the mutation pattern in a population of 40 cystinosis patients from 35 families of German and Swiss origin. CTNS mutations in 68 out of 70 alleles were identified. The common 57-kb deletion accounted for 65% of the alleles. In five patients we found a known GACT deletion at position 18-21. In two patients we identified a nucleotide substitution at codon 339 and one patient showed a CG insertion at position 697-698. In five patients we observed a G insertion at position 926-927. Moreover, five novel mutations including two deletions involving exon 3 (61-61+2delGGT) and exon 6 (280delG), two insertions in exon 6 (292-293insA) and exon 7 (684insCACTT) and one nucleotide substitution in exon 11 (923G>T) have been identified. These data provide a basis for routine molecular diagnosis of cystinosis in the central European population, especially in cystinosis patients of German and Swiss origin.

Amino Acid Sequence↗

PubMed2002

Remark on utility and error rates of the allopurinol test in detecting mild ornithine transcarbamylase deficiency.

Carriers of X-linked ornithine transcarbamylase deficiency (OTCD) are themselves mildly affected. The allopurinol test is quite sensitive (92.7%) and very specific in detecting these carriers. Consequently, it has also been recommended for the diagnosis of mild OTCD in the general population. However, there is a controversy on its utility since OTCD could not be demonstrated in several patients with positive test results but negative family histories. We show that this controversy is due to an improper use of statistical concepts, i.e., to the postulate of a specificity of "100%," and to the confusion of specificity with type I error rate. Spontaneous orotic aciduria implies a positive allopurinol test and limits the specificity of the test to a maximum of 99.7%. Therefore, according to Bayes' theorem, almost all positive test results in the general population must turn out to be type I errors, due to the minute prevalence (1/32,000) of mild OTCD (i.e., asymptomatic carriers and male patients with inapparent disease). Family history seems to be the only preselective parameter that can sufficiently raise the prevalence in the group to be tested. Bayesian analysis also yields the rate of type II errors (OTCD inspite of a negative test) which is high in closely related at-risk females (22.6% in mothers of male patients) but minimal in the general population. Conclusion. The allopurinol test is useful for the exclusion but not for the diagnosis of inapparent OTCD in sporadic individuals. Test results in possible carriers should be interpreted with caution.

Allopurinol↗

PubMed2002

The kyphoscoliotic type of Ehlers-Danlos syndrome (type VI): differential effects on the hydroxylation of lysine in collagens I and II revealed by analysis of cross-linked telopeptides from urine.

The kyphoscoliotic type of Ehlers-Danlos syndrome (EDS type VIA) (OMIM 225400) is an autosomal recessive connective tissue disorder that results from mutations in the lysyl hydroxylase 1 gene (PLOD1) causing underhydroxylation of lysine residues in tissue collagens, particularly of skin. Previous studies have shown that the pool of collagen cross-linking amino acids, hydroxylysyl pyridinoline (HP) and lysyl pyridinoline (LP) excreted in urine has an abnormally low HP/LP ratio, which is diagnostic of the condition. Here we isolated cross-linked peptides containing these residues from the urine of a child with EDS VIA homozygous for a mutation that results in a stop codon and effective null expression of PLOD1 enzyme activity. Peptides that had originated from bone type I collagen and cartilage type II collagen were identified. A cross-linked N-telopeptide fraction that is derived from bone type I collagen contained only LP, no HP, which means that the helical lysines at residues 930 of alpha 1(I) and 933 of alpha 2(I) of the collagen triple-helix had not been hydroxylated. The equivalent peptide fraction from a normal child's urine gave a ratio of HP to LP of 1.5:1 typical for normal bone collagen. A second cross-linked peptide that is derived from the C-telopeptide domain of cartilage type II collagen showed both HP and LP in a 2:1 ratio, compared with 18:1 for the equivalent peptide from a normal child's urine. The results show that in EDS VIA, bone type I collagen is more markedly underhydroxylated than cartilage type II collagen, at least at those helical sites that form cross-links. The residual fraction of HP found in the urine of EDS VI patients therefore appears to be contributed in significant part by the degradation products of cartilage. Since PLOD1 is null, other PLOD genes must be responsible for the helical hydroxylation activity that results in HP. The presented approach of analyzing urinary cross-linked C-telopeptide fragments of type II collagen may allow the detection of chondrodysplasias due to genetic defects in lysyl hydroxylase isoforms active in cartilage.

Chromatography, High Pressure Liquid↗

PubMed2002

Quality of life and psychologic adjustment in children and adolescents with early treated phenylketonuria can be normal.

OBJECTIVES: To assess quality of life and psychologic adjustment in children and adolescents with early treated phenylketonuria (PKU). STUDY DESIGN: Cross-sectional study. METHODS: Assessment of 37 patients with PKU between 3 and 18 years of age (mean, 10.9 years), with parents used as informants. The TNO-AZL Questionnaire for Children's Health-Related Quality of Life and Child Behavior Checklist scores were compared with healthy reference groups. Predictive values of sociodemographic and clinical variables for quality of life and psychologic adjustment were assessed. RESULTS: Most dimensions of quality of life in children with PKU were not different from reference values. The only deviation in the PKU group was a reduction of positive emotions. Psychologic adjustment in patients with PKU was better than in a healthy reference group. Whereas sociodemographic variables and phenylalanine levels in the 12 months preceding the study were not associated to quality of life and psychologic adjustment, a long-term negative effect of higher phenylalanine levels during the first year of life on some dimensions of quality of life and psychologic adjustment could be identified. CONCLUSIONS: Although the results may not necessarily apply to patients with PKU in other geographic or socioeconomic contexts, they demonstrate that normal health-related quality of life and good psychologic adjustment is an achievable goal in children with PKU.

Adaptation, Psychological↗

PubMed2001

The mutation spectrum of the facilitative glucose transporter gene SLC2A2 (GLUT2) in patients with Fanconi-Bickel syndrome.

We report a total of 23 novel mutations of the SLC2A2 ( GLUT2) gene in 49 patients with a clinical diagnosis of Fanconi-Bickel syndrome (FBS). Molecular genetic analysis has now been performed in more than 50% of the 109 FBS cases from 88 families that we have been able to locate world-wide since the original report in 1949. In these 49 patients, 33 different SLC2A2 mutations (9 missense, 7 nonsense, 10 frameshift, 7 splice-site) have been detected. Thus, our results confirm that mutations of SLC2A2 are the basic defect in patients with FBS. Mutations of SLC2A2 were detected in historical FBS patients in whom some of the characteristic clinical features (hepatorenal glycogen accumulation, glucose and galactose intolerance, fasting hypoglycemia, a characteristic tubular nephropathy) and the effect of therapy were described for the first time. Mutations were also found in patients with atypical clinical signs such as intestinal malabsorption, failure to thrive, the absence of hepatomegaly, or renal hyperfiltration. No single prevalent SLC2A2 mutation was responsible for a significant number of cases. In a high percentage (74%) of FBS patients, the mutation is homozygous, so we conclude that the prevalence of SLC2A2 mutations is relatively low in most populations. No mutational hot spots within SLC2A2 or even within homologous sequences among the genes for facilitative glucose transporters were detected.

Alternative Splicing↗

PubMedDate not supplied

In vitro proteoglycan sulfation derived from sulfhydryl compounds in sulfate transporter chondrodysplasias.

Mutations in a sulfate-chloride antiporter gene, the diastrophic dysplasia sulfate transporter (DTDST), have been associated with a family of skeletal dysplasias including recessive multiple epiphyseal dysplasia, diastrophic dysplasia (DTD), atelosteogenesis type 2, and achondrogenesis type 1B (ACG1B). DTDST function is crucial for uptake of extracellular sulfate required for proteoglycan (PG) sulfation; the tissue-specific expression of the clinical phenotype may be the consequence of the high rate of PG synthesis in chondrocytes and the ensuing high sulfate requirement. We have studied the contribution of cysteine and its derivatives to PG sulfation in fibroblast and chondrocyte cultures from sulfate transporter dysplasia patients. Incubation of ACG1B fibroblasts in medium containing different concentrations of cystine indicated partial recovery of PG sulfation as measured by HPLC disaccharide analysis of chondroitin sulfate PGs; similar results were observed after incubation with N-acetylcysteine. When both compounds were tested in primary chondrocytes from a DTD patient, partial rescue of PG sulfation was observed, suggesting that the metabolic pathways producing cytoplasmic sulfate from thiols are also active in this cell type.

Acetylcysteine↗