BRCA1 mutations and phenotype.

More than 100 mutations have been described for the breast-cancer-susceptibility gene BRCA1. The paper describes phenotypical aspects of three selected mutations located at the beginning, in the middle, and at the end of the gene. A remarkable decrease of the age of diagnosis of the mammary carcinoma is observed with increasing length of the putative gene product, combined with greater severity of the disease.

BRCA1 mutations in German breast-cancer families.

We analyzed germline mutations of the BRCA1 gene in 20 German breast/ovarian-cancer families. BRCA1 mutations co-segregating with breast-cancer susceptibility were identified in 3 of these families. All mutations were found to generate a premature stop codon leading to the synthesis of truncated BRCA1 proteins of different length. Nine polymorphisms were detected in BRCA1, 4 of which have not been described previously. Analysis of familial tumors for LOH revealed that only the disease-related allele of BRCA1 was present.

BRCA1 mutation update and analysis.

The discovery of the BRCA1 gene involved in the development of human hereditary breast cancer led to extensive international efforts to identify the mutations leading to the disease. The new listing covers 127 mutations published in the indicated papers before 30 April 1996; 55% of the mutations are localized in exon 11, followed by exons 2 (5.5%), 5 and 16 (4.7% each).

Identification of three novel mutations in the CFTR gene using temperature-optimized non-radioactive conditions for SSCP analysis.

Optimal temperature conditions for the detection of 28 known mutations on 15 exons of the human cystic fibrosis transmembrane conductance regulator gene by single strand conformation polymorphism analysis using the Diagen TGGE Apparatus were established. This procedure was applied to the detection of unknown mutations in 58 non-deltaF508 chromosomes. Three novel mutations, -471del3 (5' flanking region), 3171insC (exon 17a) and 4700(T)8/9 (3' non-translated region) of the CFTR gene were found. Mutation 3171insC occurred in conjunction with the delta F508 mutation on the other allele of a child presenting with severe pathology. Mutation -471del3 has so far only been found in one healthy individual and her father, and 4700(T)8/9 is a DNA sequence polymorphism.

Acceptability of carrier screening for cystic fibrosis during pregnancy in a German population.

A pilot project offering voluntary heterozygote screening for the delta F508 mutation causing cystic fibrosis (CF) to 638 pregnant women attending two antenatal clinics in the eastern part of Berlin was carried out from 1990-1993. Participation was invited using an information leaflet and inclusion in the study was conditional on written informed consent. Of those invited to participate, only one refused to be tested, on the grounds of non-acceptance of prenatal diagnosis. Eighteen pregnant women were identified as carriers of the delta F508 mutation. All of them and their male partners accepted counselling in which the genetics of CF, its prognosis and treatment were explained, with emphasis on the meaning of heterozygosity, the fact that carriers are healthy, and the risk of an affected fetus when only one parent is identified as a heterozygote. All partners agreed to be tested for the delta F508 R553X and G551D mutations and a second counselling session was carried out after this test result was available. No problems were observed during initial testing but, as in other studies, we found considerable anxiety on being given the result in all couples where the woman tested positive; this was reduced substantially by counselling and when the partner tested negative. All probands found to be carriers stated that they found screening acceptable. In contrast to the cautious statement by the German Berufsverband Medizinische Genetik and the hostile reaction from a representative of the CF self-support organisation towards community-based heterozygote screening for CF, this study shows that CF screening is generally acceptable in this German population and that it is actively taken up by most pregnant women when offered.

A 32-bp deletion (2991del32) in the cystic fibrosis gene associated with CFTR mRNA reduction.

Cystic fibrosis, a common recessive disorder of exocrine glands, is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. We describe the identification of a 32-bp deletion within the coding region of CFTR that involves the nucleotides 2991-3022 in exon 15 (2991del32). This unusual frameshift mutation was confirmed in three unrelated German families, accounting for a frequency of 0.3% in 1,028 CF chromosomes. All identified patients are compound heterozygotes for 2991del32 and for the most frequent cystic fibrosis mutation, delta F508. The evaluation of clinical data revealed typical symptoms of cystic fibrosis, including pancreatic insufficiency, in all three index cases. To characterize further the mutation in the CFTR transcript, we analysed RNA from lymphocytes by reverse transcription and PCR amplification. 2991del32 transcripts were detectable neither in the RNA sample from a patient compound heterozygous delta F508/2991del32 nor in the parental sample heterozygous wild-type/2991del32. These data indicate that the 32-bp deletion causes a pancreas insufficient cystic fibrosis phenotype by a severe reduction of CFTR mRNA.

Non-isotopic analysis of single strand conformation polymorphism (SSCP) in the exon 13 region of the human dystrophin gene.

More than 30% of Duchenne and Becker muscular dystrophy (DMD/BMD) patients have no gross DNA rearrangements like deletions or duplications. The large size of the coding sequence of the dystrophin gene (11 kilobases) complicates systematic identification of point mutations. Recently reported approaches based on genomic DNA or mRNA show that chemical cleavage of mismatches is an effective but time consuming and technically demanding method for the identification of point mutations in the human dystrophin gene. We have used a fast and convenient system consisting of PCR amplification of genomic DNA, non-isotopic SSCP analysis, and direct sequencing of PCR products for the detection of mutations in exon 13 and adjacent intron sequences. Sixty-eight DMD patients without detectable deletions or duplications were analysed, resulting in the identification of a point mutation in the coding sequence and two polymorphisms in the 5' flanking intron. The C to T change of the first nucleotide in the third triplet leads to a stop codon and seems to be the cause of the functional deficiency of the gene product in this patient.

Myoelectric motility patterns following open versus laparoscopic cholecystectomy.

Laparoscopic surgery is associated with a lack of postoperative ileus. To determine if differences exist in postoperative motility patterns, 8 dogs were instrumented with bipolar electrodes 10-14 days prior to open (n = 4) or laparoscopic (n = 4) cholecystectomy. In both groups, Phase II activity disappeared in the first 24 h after operation. The appearance of the migrating myoelectric complex in the small intestine and the migrating colonic complex were used as criteria for recovery from postoperative ileus. Postoperative migrating myoelectric complex cycle length, migrating myoelectric complex, migration velocity, and colonic spike bursts/hour were also measured. No statistically significant differences were observed between groups in study parameters examined. Postoperative myoelectric motility patterns in dogs undergoing open versus laparoscopic cholecystectomy are not different. Other factors may be responsible for the rapid return to oral intake following laparoscopic cholecystectomy.

[Molecular biological analysis of cystic fibrosis--a model example for the strategy of "reverse genetics"]. / Die molekularbiologische Analyse der Cystischen Fibrose--Ein Modellbeispiel für die Strategie der "Reversen Genetik".

The elucidation of the basic defect causing cystic fibrosis (CF) is a paradigm for the application of "reverse genetics" to the analysis of human genetic disease. Following this strategy, linkage analysis localized the responsible gene for CF on chromosome 7. Chromosome mediated gene transfer and chromosome walking and jumping led to the isolation of the CFTR-gene and its cDNA. A major 3 bp deletion mutation (DeltaF508) and more than 100 other mutations of this gene have been identified as molecular basis of cystic fibrosis. The CFTR-amino acid sequence, obtained by conversion of the cDNA-sequence, indicates that CFTR belongs to a group of integral membrane transport proteins (ABC-proteins). The normal cAMP-stimulated chloride-transport, lacking in CF-cells is restored by transfer and expression of CFTR-cDNA-recombinants in these cells. CFTR is most likely itself a chloride channel. The molecular identification of this gene has already led to substantial advances in diagnosis and prevention of this disease. New therapeutic approaches by pharmacological means or gene therapy are expected from the further molecular and functional analysis of the CFTR-gene.

Prevalence of cystic fibrosis mutations in the East German population.

A representative multicenter cystic fibrosis (CF) mutation analysis on about half of all known cystic fibrosis patients of the 5 East German Länder is reported. Analyses for 17 mutations, among them Delta F508, R553X, G542X, S549R,N,I, G551D, S1255X, R347P,H, and Y122X, were performed. As expected, the delta F508 mutation in exon 10 of the CFTR gene is the major gene alteration causing CF in our patients. However, in comparison to studies from Western Germany, a significantly lower percentage of just over 60% is found in our patients, resembling data obtained from slavonic populations. The severe phenotype of cystic fibrosis is most frequently associated with homozygosity for the delta F508 mutation. No particular allele association could be found with the intermediate and mild phenotypes of this disease. The next most frequent of the investigated mutations is R553X (13.3% of non-delta F chromosomes) followed by R347P (9.2%) and G542X (4.4%).

A pooling strategy for heterozygote screening of the delta F508 cystic fibrosis mutation.

A theoretical and practical approach to economize the analysis of large DNA sample numbers for identifying heterozygosity of the delta F508 mutation causing cystic fibrosis is presented. Sample pooling can reduce the number of polymerase chain reaction (PCR) tests for this mutation by up to 77%. Based on a mathematical model, the optimal number (n) of samples to be united in one pool is 24 for a German population with a delta F508 heterozygosity incidence of about 1/35. We show that the PCR method is sufficient to detect one heterozygote for the delta F508 mutation in a pool of up to 49 non-delated DNA samples.

Deletion analysis of DMD/BMD families from the German Democratic Republic and selected regions of Czechoslovakia and Hungary.

Over the last two years we have screened 183 DMD/BMD families requesting prenatal diagnosis. Using cDNA probes cf56a,b we have detected exon deletions in 72 of them. In 62 cases the deletion was also detectable with currently available PCR primers. Deletion analysis for exons 8, 17, and 19, using either PCR or Southern blotting techniques, was performed for 65 of the 111 families which showed no deletions with cf56a,b. Eight of them were deleted for one or more of these exons. PCR offers new possibilities for deletion analysis in families without a living patient using either Guthrie papers or histologically conserved material from the dead patient. In 20 of 25 patients, we observed concordance between the clinical picture and the molecular deletion analysis in accordance with the open reading frame hypothesis. Five patients, however, presented with DMD in spite of our analysis showing an in frame deletion. Carrier determination in families in which DMD is caused by a deletion using linkage, dosage, or breakpoint analysis is discussed.

First analysis of the F508 deletion in cystic fibrosis patients from the GDR.

Cystic fibrosis (CF) patients (n = 157) from the GDR were analysed for the occurrence of the recently discovered 3bp deletion causing CF. About 50% of all investigated patients were homozygotes and about 30% heterozygotes for this deletion. Of the analysed CF chromosomes from these patients, 62% carry the deletion, which is in strong linkage disequilibrium with the KM19 restriction fragment length polymorphism allele 2 and the 1/2 XV2c/KM19 haplotype.

Possibilities and limitation of prenatal diagnosis and carrier determination for Duchenne and Becker muscular dystrophy using cDNA probes.

Two cDNA probes, cf23a and cf56a, identify deletions of selected exons in about 50% of our DMD/BMD patients. We have estimated the most likely order of the 11 exons detectable with both probes with respect to the different extensions of the deletions. In one of our BMD pedigrees, the observed deletion could be traced in the affected males through three generations. This result shows that with the use of cDNA probes detecting deletions, the only risk of error in genomic prenatal diagnosis is the general high frequency of new mutations for DMD/BMD. This is important progress in diagnosis compared to the 2 to 5% risk of misdiagnosis because of crossing over events using conventional linkage analysis with bridging or intragenic probes. The first prenatal diagnosis of an unaffected fetus of a woman who is a DMD carrier according to ultrasound examination is described. In one of our DMD males, the cDNA probe cf56a detects a deletion breakpoint. His sister also shows the altered band and is therefore a DMD carrier, while his mother has a totally normal band pattern. The interpretation of this observation could be either germline mosaicism or two identical new mutations. The identification of deletion breakpoints is a new diagnostic strategy, especially for carrier determination, which excludes misdiagnosis owing to crossing over events and the problems of dosage estimation. It is, however, limited by the low frequency of breakpoints detectable with cDNA probes. Therefore, the generation of new intron probes in this region is an important goal.