Manfred Schlösser

Detection of a novel point mutation causing haemophilia A by PCR/direct sequencing of ectopically-transcribed factor VIII mRNA

SummarySpecifically-primed reverse transcripts of lymphocyte-derived factor VIII (FVIII) mRNA were successfully amplified by means of the polymerase chain reaction (PCR) thus further extending the phenomenon of ectopic (“illegitimate”) transcription of tissue-specific genes. The diagnostic potential of a basal rate of transcription in non-expressing tissues was then demonstrated by the detection of a novel point mutation in the FVIII gene causing haemophilia A by PCR/direct sequencing of ectopically transcribed mRNA derived from patient lymphocytes.

Five novel mutations in the L1CAM gene in families with X linked hydrocephalus.

Five novel mutations have been identified in the gene encoding L1CAM, a neural cell adhesion protein, in families with X linked hydrocephalus (XHC). Interestingly, all five mutations are in the evolutionarily highly conserved Ig-like domains of the protein. The two frameshift mutations (52insC and 955delG) and the nonsense mutation (Trp276Ter) most probably result in functional null alleles and complete absence of L1CAM at the cell surface. The two missense mutations (Tyr194Cys and Pro240Leu) may considerably alter the structure of the L1CAM protein. These data provide convincing evidence that XHC is genetically extremely heterogeneous.

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.

Distribution patterns of the ΔF508 mutation in the CFTR gene on CF-linked marker haplotypes in the German population

SummaryWe have measured the frequency of the ΔF508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and its association with cystic fibrosis (CF)-linked marker haplotypes in the German population. Based on the analysis of 400 CF chromosomes, the frequency of the ΔF508 mutation is estimated to be 77.3%, the vast majority being associated with marker haplotype KM19-XV2c 2 1. Our data further suggest the presence of another frequent CF mutation associated with this marker haplotype.

Single-strand conformation polymorphism (SSCP) analysis of exon 11 of the CFTR gene reliably detects more than one third of non-ΔF508 mutations in German cystic fibrosis patients

SummaryIn Central Europe, the ΔF508 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-ΔF508 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-ΔF508 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.

The bovine protamine 2 gene: Evidence for alternative splicing

Protamine 2 (PRM2) is a low molecular weight arginine-rich protein which is present in haploid spermatogenic cells of human and mouse. Although the bull PRM2 gene is translated and transcribed at low levels, the protein could not be detected. The gene was isolated from a cosmid library and was found to consist of two exons (298 and 50 bp, respectively) interrupted by an intron of 142 bp. As compared to the PRM2 genes of man, mouse and rat the bovine gene lacks a highly conserved sequence coding for the amino acids RLHRIH. Furthermore, primer extension experiments on bull PRM2 mRNA and sequencing of junction fragments revealed alternative splicing of mRNA resulting in two putative isoforms of the protein. The most abundant transcript is spliced at the conserved splice donor site found in exon 1 at position 236 giving rise to an in-frame deletion of 63 bp as compared to the cDNA sequence (Maier et al. (1990) Nucleic Acids Res. 18, 1249-1254). The less abundant longer mRNA was not detectable by radioactive primer extension. The corresponding cDNA was obtained by performing PCR with reverse transcribed bull testis RNA or with a spermatid specific cDNA library. Alternative splicing should result in an addition of 21 nonpolar amino acids in the derived polypeptide and an altered protein conformation and function.