Biomedical subjects
J Reiss
Publications and source records attributed to J Reiss.
Two cystic fibrosis patients with the genotype G542X/G551D.
Two adult sisters affected by cystic fibrosis were both shown to carry two different alterations within exon 11 of the CFTR gene, the nonsense mutation G542X and the missense mutation G551D. Both patients exhibit a relatively benign clinical course. In the described patients, G542X functions as a "mild" allele and is, in this respect, dominant to the "severe" G551D.
Miso from peas (Pisum sativum) and beans (Phaseolus vulgaris) of domestic origin. Fermented foods from agricultural products in Europe. II.
Miso is a fermented soybean paste widely used in Japan as a soup base or as a seasoning agent. Koji (cereal grains with the mold Aspergillus oryzae) serves as enzyme source. Peas (Pisum sativum) and beans (Phaseolus vulgaris) of German origin can be used as substitutes for soybeans in the preparation of miso-like products. The legumes (peas, beans and soybeans for comparison) are washed, soaked in boiled water, dehulled and cooked for 35 min at reduced pressure. After grounding the seeds are mixed with salt, koji and mugi miso as starter and incubated at 45 degrees C for 14 days. During fermentation the glucose content increases up to 8-10 days and subsequently drops down. Crude protein decreases during incubation while dry matter increases. The pH value of all three miso types decreases during the fermentation period. Most of the 40 test persons characterize odor and flavor of the three misos as aromatic or sour; pea miso is often recorded to have a sweet-like odor and flavor. A legume-like taste of the final products has not been recorded.
Use of a sealed minichamber for direct observation and evaluation of the in vitro effect of cigarette smoke on sperm motility.
OBJECTIVE: To determine the in vitro effect of cigarette smoke on sperm motility and survival in an attempt to find a possible association with clinical studies that had pointed to the existence of such an effect in vivo. DESIGN: Laboratory experiments in which the effect of cigarette smoke on human sperm placed in a sealed minichamber was directly observed and determined by motion analysis. PATIENTS: High-quality fresh semen specimens were obtained from healthy donors and used for the experimental studies. INTERVENTIONS: Various doses of filtered and nonfiltered cigarette smoke, collected by an improvised manual smoking machine, were streamed at a rate of 2 mL/s into sealed chambers containing samples of washed spermatozoa. Evaluation of the changes in sperm motility was performed in real time by direct microscopical observation and repeated determinations by the multiple exposure photography technique until their complete immobilization. RESULTS: Streaming of 100 mL of cigarette smoke induced a dramatic drop in sperm motility, which caused sperm immobilization in about 15 minutes. This effect showed a clear dose-response relationship either with the amounts streamed or with the time during which sperm were exposed to cigarette smoke. The harmful effect was almost the same whether sperm were exposed to filtered or nonfiltered cigarette smoke. The role of carbon monoxide and hydrogen cyanide on these findings was studied separately, and it was shown that the inhibitory effect was not due to the presence of these two important components of cigarette smoke. CONCLUSIONS: The clear evidence of the in vitro harmful effect of crude cigarette smoke on sperm motility points to the possibility that in heavy smokers certain toxic agents may reach mature spermatozoa or their precursors via the blood stream and induce their harmful effect on sperm motility in vivo.
A comprehensive CFTR mutation analysis of German cystic fibrosis patients.
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CFTR transcripts are undetectable in lymphocytes and respiratory epithelial cells of a CF patient homozygous for the nonsense mutation R553X.
In order to analyse the influence of the nonsense mutation R553X on CFTR gene expression, transcripts from epithelial cells and lymphocytes were examined from nine subjects (one CF patient homozygous for R553X, one CF patient compound heterozygous for R553X/delta F508, four CF carriers heterozygous for R553X, one CF carrier with the genotype delta F508/N, and two uncharacterized normal adults). After reverse transcription of the region from exons 10 to 13 to cDNA, fragments of the expected size were amplified from all heterozygous and normal subjects. In three subjects an additional alternatively spliced product was observed, which was found to contain a termination codon. In repeated experiments it was not possible to detect any CFTR mRNA in cells derived from the R553X homozygous patient. Furthermore, in subjects heterozygous for R553X we could not detect by hybridisation with a specific oligonucleotide probe and direct sequencing any CFTR mRNA derived from the R553X allele. However, the wild type product was present in all of these subjects. Our results support the view that nonsense mutations in the CFTR gene can lead to a reduction or absence of cytoplasmic CFTR mRNA.
Single-strand conformation polymorphism (SSCP) analysis of exon 11 of the CFTR gene reliably detects more than one third of non-delta F508 mutations in German cystic fibrosis patients.
In Central Europe, the delta F508 deletion accounts for approximately 75% of mutations in the cystic fibrosis transmembrane conductance regulator gene causing cystic fibrosis. The remainder comprise a large number of individually infrequent mutations whose detection requires a disproportionately large effort. However, a sizeable proportion of non-delta F508 mutations have been found to cluster within exon 11. We have taken advantage of this clustering to detect a total of five previously described point mutations present on 26/72 (36%) non-delta F508 chromosomes by polymerase chain reaction/direct sequencing of exon 11. These exon 11 mutations were then subjected to single-strand conformation polymorphism (SSCP) analysis, which was shown (i) to discriminate reliably between mutant and wildtype alleles and (ii) to generate reproducible mutation-specific band patterns. This analysis thus represents the first attempt to assess SSCP analysis retrospectively, and serves to illustrate the potential of this screening technique in diagnostic medicine.
A novel CFTR mutation, 4035delA, detected by non-radioactive SSCP analysis.
German cystic fibrosis patients were screened for mutations in exon 21 of the cystic fibrosis transmembrane conductance regulator gene by a non-radioactive variation of the single-strand conformation polymorphism technique. Asymmetric polymerase chain reaction amplification was used to produce single strands of exon-containing genomic sequences that were analyzed on polyacrylamide gels subsequently stained with ethidium bromide. This rapid technique led to the identification of a novel mutation, a 1-bp deletion at position 4035(A) of the cDNA sequence. The patient, who is also heterozygous for the delta F508 mutation, exhibits an intermediate form of the disease.
Omission of exon 12 in cystic fibrosis transmembrane conductance regulator (CFTR) gene transcripts.
Cystic fibrosis transmembrane conductance regulator (CFTR) mRNA transcripts isolated from both expressing and "non-expressing" cell types of normal individuals exhibit differential splicing to a variable extent in a region encoding the putative nucleotide binding fold of the CFTR polypeptide. Sequence analysis of the aberrant fragments obtained after cDNA polymerase chain reaction amplification confirmed the in-frame joining of exons 11 and 13. The proportion of alternative splicing is reproducible and constant in a given individual. The omission of exon 12 in a significant proportion of transcripts supports the hypothesis that a minimal amount of correctly expressed CFTR is sufficient for the maintenance of a clinically normal phenotype.
Ornithine transcarbamylase (OTC) deficiency in a female patient with a de nova deletion of the paternal X chromosome.
A girl with ornithine transcarbamylase (OTC) deficiency was investigated for molecular and cytogenetic abnormalities that might explain this phenotype. Analysis with polymorphic DNA markers indicated that the patient did not inherit paternal alleles of the OTC locus, but that she did inherit the proximal locus DXS7 and the long arm of chromosome X. High-resolution cytogenetic analysis of the patient indicated a deletion of Xp11.4-p21, whereas both parents had normal karyotypes. Since the mother might be heterozygous according to biochemical tests, a second mutation within the maternal OTC gene cannot be excluded.
Incidence and expression of the N1303K mutation of the cystic fibrosis (CFTR) gene.
The N1303K mutation was identified in the second nucleotide binding fold of the cystic fibrosis (CF) gene last year. We have gathered data from laboratories throughout Europe and the United States of America in order to estimate its frequency and to attempt to characterise the clinical manifestations of this mutation. N1303K, identified on 216 of nearly 15,000 CF chromosomes tested, accounts for 1.5% of all CF chromosomes. The frequency of the N1303K allele varies significantly between countries and ethnic groups, being more common in Southern than in Northern Europe. This variation is independent of the delta F508 allele. It was not found on UK Asian, American Black or Australian chromosomes. N1303K is associated with four different linked marker haplotypes for the polymorphic markers XV-2c, KM.19 and pMP6d-9. Ten patients are homozygous for this mutation, whereas 106 of the remainder carry one of 12 known CF mutations in the other CF allele. We classify N1303K as a "severe" mutation with respect to the pancreas, but can find no correlation between this mutation, in either the homozygous or heterozygous state, and the severity of lung disease.
De novo splice site mutation in the antithrombin III (AT3) gene causing recurrent venous thrombosis: demonstration of exon skipping by ectopic transcript analysis.
A single basepair substitution at conserved position -1 in the exon 3a donor splice site of the liver-expressed antithrombin III (AT3) gene was detected by PCR/direct sequencing in a patient with sporadic type 1 ATIII deficiency and recurrent venous thrombosis. The lesion, a heterozygous silent AAG----AAA transition at Lys 176 occurred de novo in the proposita. Ectopic transcript analysis of lymphocyte mRNA demonstrated the presence of an abnormally sized mRNA specific to the patient which was shown by cDNA sequencing to lack exon 3a. Oligonucleotide discriminant hybridization demonstrated the absence of any detectable transcript of normal length derived from the disease allele. These findings demonstrate the utility of ectopic transcript analysis in the characterization of defects of mRNA splicing.
Simple non-radioactive detection of the CFTR mutation N1303K by artificial creation of a restriction site.
N1303K is one of the most frequent non-delta F508 mutations causing cystic fibrosis in Central Europe. Since no restriction site is altered by this mutation and no other frequent mutations are known so far in exon 21, the detection requires a separate and laborious test. A mismatched primer was used to create an artificial Hin dIII site in amplified wildtype DNA, which is destroyed by the mutation. This allows for rapid and convenient detection by restriction enzyme digestion.
Identification of a new DMD gene deletion by ectopic transcript analysis.
The detailed genetic analysis of the Duchenne/Becker muscular dystrophy gene is hindered by the large number of exons involved and their separation by huge introns. These problems can be overcome by the analysis of mRNA rather than genomic DNA and ectopic transcripts derived from peripheral blood lymphocytes provide a convenient source of material. Using reverse transcription and nested PCR, we show here a comprehensive strategy for the rapid and complete analysis of the coding sequences from complex genes and illustrate its potential by the identification of a hitherto undescribed single exon deletion.
[Use of polymerase chain reaction (PCR) in clinical diagnosis].
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The frequency of mutations in exon 11 of the CF gene in Polish cystic fibrosis patients.
Results of mutation analysis in exon 11 of the CF gene have been presented. Using the SSCP technique 18 mutations (of four different types) were detected in cystic fibrosis patients of Polish origin. Thus, we were able to detect in exon 11 about 10% of all CF mutations occurring in the affected population examined.
Discrimination between recurrent mutation and identity by descent: application to point mutations in exon 11 of the cystic fibrosis (CFTR) gene.
A total of 75 non-delta F508 chromosomes from 59 German cystic fibrosis patients was screened for mutations in exon 11 of the cystic fibrosis (CFTR) gene. These Caucasian patients were found to possess an identical haplotype background for two common mutations (G551D, R553X) consistent with their being identical by descent. However, a different R553X associated haplotype found in American black patients was suggestive of recurrent mutation, a postulate supported by the location of the R553X alteration in a hypermutable CpG dinucleotide. Likelihood estimates for recurrent mutation and identity by descent were compared and strongly supported the hypothesis of recurrent R553X mutation. The ability to distinguish between these two alternatives provides an indication of whether or not the search for mutations should be restricted to chromosomes with similar haplotypes.
Detection of human spermatid-specific transcripts in peripheral blood lymphocytes of males and females.
We describe the detection of ectopic ("illegitimate") transcripts of the proacrosin and protamine 2 genes, which are specific for human spermatogenesis, in non-cultured peripheral blood lymphocytes. After specifically-primed reverse transcription of total lymphocyte RNA, these rare transcripts can be directly visualised after two rounds of polymerase chain reaction with nested primers. Sequence and restriction analyses of the corresponding fragments have confirmed that transcripts of proacrosin and protamine 2 are present in the lymphocytes not only of males, but also of adult females.