Notes: Abstract The methylation status of 12 CpG sites in three exons of the human factor VIII (F8C) gene was examined by bisulphite genomic sequencing of human sperm DNA from 14 European Caucasians and Asians. Different CpG sites were found to vary in their methylation status both within and between individuals. Strand differences in methylation status were also detected at certain sites, a finding that could reflect hemi-methylation. No evidence for systematic deviations in methylation status were found between the two ethnic groups. Only a limited correlation was observed between the level of methylation of specific CpG sites in sperm DNA and their mutability, a finding that is probably attributable to the pattern of methylation observed in mature spermatocytes not being representative of that of the germline.

Notes: Summary This study analyses distribution patterns of the ΔF508 mutation of the cystic fibrosis transmembrane conductance regulator gene (CFTR) gene and the cystic fibrosis (CF)-linked marker loci MET, D7S23, D7S399, and D7S8 in a sample of 167 (116 complete) CF families from Bohemia and Moravia (Czechoslovakia). DNA typing was performed by polymerase chain reaction amplification, restriction analysis, and agarose or polyacrylamide gel electrophoresis. The frequency of the ΔF508 mutation in this sample is 67% and the frequency of the B haplotype is 77.6% on CF chromosomes. Linkage disequilibrium was found between ΔF508 and all markers tested.

Notes: Summary A total of 75 non-ΔF508 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) constistent 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.

Notes: Summary In order to facilitate the screening for the less common mutations in the cystic fibrosis (CF) gene viz., the CF transmembrane conductance regulator gene (CFTR), marker haplotypes were determined for German nonCF (N) and CF chromosomes by polymerase chain reaction analysis of four polymorphisms upstream of the CF gene (XV-2c, KM.19, MP6-D9, J44) and six intragenic polymorphisms (GATT, TUB9, M470V, T854T, TUB18, TUB20) that span the CFTR gene from exon 6 through exon 21. Novel informative sequence variants of CFTR were detected in front of exons 10 (1525-61 A or G), 19 (3601-65 C or A), and 21 (4006-200 A or G). The CF locus exhibits strong long-range marker-marker linkage disequilibrium with breakpoints of recombination between XV-2c and KM.19, and between exons 10 and 19 of CFTR. Marker alleles of GATT-TUB9 and TUB18-TUB20 were found to be in absolute linkage disequilibrium. Four major haplotypes encompass more than 90% of German N and CF chromosomes. Fifteen CFTR mutations detected on 421 out of 500 CF chromosomes were each identified on one of these four predominant 7-marker haplotypes. Whereas all analysed ΔF508 chromosomes carried the same KM.19-D9-J44-GATT-TUB9-M470V-T854T haplotype, another frequent mutation in Germany, R553X, was identified on two different major haplotypes. Hence, a priori haplotyping cannot exclude a particular CF mutation, but in combination with population genetic data, enables mutations to be ranked by decreasing probability.

Notes: Abstract A multi-domain molecular model of factor IXa was constructed by comparative methods. The quaternary structure of the protein was assembled by docking individual domains through consideration of their shape complementarity, polaric properties and the location of cross-reacting material positive/negative (CRM+/−) variants on domain surfaces. Some 217 different missense mutations in the factor IX (F9) gene were then selected for study. Using maximum likelihood analysis, missense mutations affecting highly conserved amino acid residues of factor IX were shown to be 15–20 times more likely to result in haemophilia B than those affecting non-conserved residues. However, about one quarter of this increase in likelihood of clinical observation could be attributed to the magnitude of the amino acid exchange. Missense mutations in structurally conserved residues were found to be 2.1-fold more likely to come to clinical attention than those in structurally variable residues. Missense mutations in residues whose side chains were inwardly pointing were 3.6-fold more likely to be observed than those in surface residues. These observations imply a complex hierarchy of sequence/structure conservation in the protein. The severity of the clinical phenotype correlated with both the extent of the evolutionary sequence conservation of the residue at the site of mutation and the magnitude of the amino acid exchange. Further, the substitution of residues exhibiting minimal side chain solvent accessibility was associated disproportionately with severe haemophilia compared with that of surface residues. Clusters of CRM+ mutations were observed at factor IX-specific residues on the surface of the molecule. These clusters may reflect factor IX-specific docking interactions. The likelihood that a given factor IX mutation will come to clinical attention is therefore a complex function of the sequence characteristics of the F9 gene, the nature of the amino acid substitution, its precise location and immediate environment within the protein molecule, and its resulting effects on the structure and function of the protein.

Notes: Abstract We describe the detection of a novel missense mutation (Thr176→Ile) that is located at the neo N-terminus of activated protein C. The Thr176→Ile substitution leads to a type 1 deficiency state. Evidence is presented suggesting that this residue plays a role in pivoting the N-terminus of protein C to fold into the oxyanion hole.

Notes: Summary We 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.

Notes: Summary Reports describing short (〈 20 bp) gene deletions causing human genetic disease were collated in order to study underlying causative mechanisms. Deletion break-point junction regions were found to be non-random both at the nucleotide and dinucleotide sequence levels, an observation consistent with an endogenous sequencedirected mechanism of mutagenesis. Direct repeats of between 2 bp and 8 bp were found in the immediate vicinity of all but one of the 60 deletions analysed. Direct repeats are a feature of a number of recombination, replication or repair-based models of deletion mutagenesis and the possible contribution of each to the spectrum of mutations examined was assessed. The influence of parameters such as repeat length and lenght of DNA between repeats was studied in relation to the frequency, location and extent of these deletions. Findings were broadly consistent with a slipped mispairing model but the predicted deletion of one whole repeat copy was found only rarely. A modified version of the slipped mispairing hypothesis was therefore proposed and was shown to possess considerable explanatory value for ∼ 25% of deletions examined. Whereas the frequency of inverted repeats in the vicinity of gene deletions was not significantly elevated, these elements may nevertheless promote instability by facilitating the formation of secondary structure intermediates. A significant excess of symmetrical sequence elements was however found at sites of single base deletions. A new model to explain the involvement of symmetric elements in frameshift mutagenesis was devised, which successfully accounted for a majority of the single base deletions examined. In general, the loss of one or a few base pairs of DNA was found to be more compatible with a replication-based model of mutagenesis than with a recombination or repair hypothesis. Seven hitherto unrecognized hotspots for deletion were noted in five genes (AT3, F8, HBA, HBB and HPRT). Considerable sequence homology was found between these different sites, and a consensus sequence (TGA/GA/GG/ TA/C) was drawn up. Sequences fitting this consensus (i) were noted in the immediate vicinity of 41% of the other (sporadic) gene deletions, (ii) were found frequently at sites of spontaneous deletion in the hamster APRT gene, (iii) were found to be associated with many larger human gene deletions/translocations, (iv) act as arrest sites for human polymerase a during DNA replication and (v) have been shown by in vitro studies of human polymerase a to be especially prone to frameshift mutation. It is proposed that dissociation of polymerase a at arrest sites may, by providing a stable single stranded substrate, lead to deletion of a DNA sequence either by slipped mispairing via a number of different secondary structure intermediates, or by strand-switching or base misincorporation. Human gene deletions thus appear to be caused by multiple mechanisms whose relative importance is probably governed by local primary and secondary DNA structure. Our ability to predict precisely the location and extent of a gene deletion is however hampered both by this complexity and by the possibility that these mechanisms may often act in combination.

Notes: Summary A total of 101 different examples of point mutations, which lie in the vicinity of mRNA splice junctions, and which have been held to be responsible for a human genetic disease by altering the accuracy of efficiency of mRNA splicing, have been collated. These data comprise 62 mutations at 5′ splice sites, 26 at 3′ splice sites and 13 that result in the creation of novel splice sites. It is estimated that up to 15% of all point mutations causing human genetic disease result in an mRNA splicing defect. Of the 5′ splice site mutations, 60% involved the invariant GT dinucleotide; mutations were found to be non-randomly distributed with an excess over expectation at positions +1 and +2, and apparent deficiencies at positions −1 and −2. Of the 3′ splice site mutations, 87% involved the invariant AG dinucleotide; an excess of mutations over expectation was noted at position -2. This non-randomness of mutation reflects the evolutionary conservation apparent in splice site consensus sequences drawn up previously from primate genes, and is most probably attributable to detection bias resulting from the differing phenotypic severity of specific lesions. The spectrum of point mutations was also drastically skewed: purines were significantly overrepresented as substituting nucleotides, perhaps because of steric hindrance (e.g. in U1 snRNA binding at 5′ splice sites). Furthermore, splice sites affected by point mutations resulting in human genetic disease were markedly different from the splice site consensus sequences. When similarity was quantified by a ‘consensus value’, both extremely low and extremely high values were notably absent from the wild-type sequences of the mutated splice sites. Splice sites of intermediate similarity to the consensus sequence may thus be more prone to the deleterious effects of mutation. Regarding the phenotypic effects of mutations on mRNA splicing, exon skipping occurred more frequently than cryptic splice site usage. Evidence is presented that indicates that, at least for 5′ splice site mutations, cryptic splice site usage is favoured under conditions where (1) a number of such sites are present in the immediate vicinity and (2) these sites exhibit sufficient homology to the splice site consensus sequence for them to be able to compete successfully with the mutated splice site. The novel concept of a “potential for cryptic splice site usage” value was introduced in order to quantify these characteristics, and to predict the relative proportion of exon skipping vs cryptic splice site utilization consequent to the introduction of a mutation at a normal splice site.