Biomedical subjects
Publications and source records attributed to D N Cooper.
A novel homozygous CCC----CTC (Pro 247----Leu) substitution was detected in the protein C genes of a patient, born to consanguineous parents, with inherited type 1 protein C deficiency and recurrent venous thrombosis. Since one of four heterozygous relatives was also clinically affected, the condition appears to be inherited as an incompletely recessive trait in this family.
Non-identical missense mutations were identified at Arg 178 in the protein C genes of two patients with heterozygous type 1 protein C deficiency and recurrent venous thrombosis.
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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.
The advent of recombinant DNA technology has contributed enormously to our understanding of human genome pathology. In this review, current approaches to the analysis and diagnosis of human genetic disease are presented and their contribution to diagnostic medicine assessed. At the level of the gene, the nonrandom nature of human gene mutation is described and the role of the local DNA sequence environment explored.
Mutations in gene promoter/regulatory regions represent an important class of lesion causing human genetic disease. Such mutations are associated with either increases or decreases in transcriptional activity mediated by the altered binding behaviour of trans-acting protein factors to specific DNA sequences in the promoter region. Although most promoter mutations are individually very infrequent, some occur at polymorphic frequencies. Both categories of lesion are likely to be important in clinical medicine and their study has already led to new insights into the mechanisms underlying the regulation of human genes.
Mutations at the factor VIII gene locus causing Haemophilia A have now been identified in many patients from many ethnic groups. Earlier studies used biased methods which detected repetitive mutations at a few CG dinucleotides. More recently rapid gene scanning methods have uncovered an extreme diversity of mutations. Over 80 different point mutations, 6 insertions, 7 small deletions, and 60 large deletions have been characterised. Repetitive mutation has been proved for at least 16 CpG sites. All nonsense mutations cause severe disease. Most missense mutations appear to cause instability of the protein, but some are associated with production of dysfunctional factor VIII molecules, thereby localising functionally critical regions of the cofactor. Variable phenotype has been observed in association with three of the latter class of genotype. This catalogue of gene lesions in Haemophilia A will be updated annually.
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Eight unrelated patients with recurrent thromboembolism, a family history of thrombosis, and plasma antithrombin III (ATIII) activity/antigen levels consistent with a diagnosis of heterozygous type I ATIII deficiency were studied by polymerase chain reaction/direct sequencing of ATIII gene exon-coding regions. Frameshift mutations of one base and two bases, respectively, were found to have occurred in two unrelated patients at the same GAG codon (Glu 245) within exon 4 of the ATIII gene. A literature search showed six further hitherto unrecognized deletion "hotspots" in four other human genes. These deletion-prone sites exhibited sufficient sequence homology with each other to derive a consensus sequence (T G A/G A/G G A/C), suggesting that deletion in human genes may not only be non-random but also sequence-directed.
Factor XI deficiency is a rare bleeding diathesis found predominantly in Ashkenazi Jewish kindreds. A recent study of six Jewish patients identified three distinct mutations (Types I, II, and III) in the factor XI gene that were sufficient to fully define the genotypes of the patients. We have investigated 63 patients with factor XI deficiency and find overall allele frequencies of 44% for the type II mutation, 31% for the type III mutation, and 0% for the type I mutation. Therefore, 25% of the mutant factor XI alleles in our sample remain undefined. However, the distribution of mutant alleles is significantly different between Jewish and non-Jewish populations with hitherto undefined mutations accounting for 84% of the disease alleles in non-Jewish patients. Plasma factor XI:C levels were found to differ significantly between different homozygous and compound heterozygous genotypes and the inheritance of the II/III genotype was found to carry an increased risk of the most severe bleeding tendency.
An updated list of DNA sequences cloned from the human genome over the past year (1990) is presented. Intended as a guide to clone availability, this list includes published reports of cDNA, genomic and synthetic clones comprising both gene and pseudogene sequences.
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Reports describing short (less than 20 bp) gene deletions causing human genetic disease were collated in order to study underlying causative mechanisms. Deletion breakpoint junction regions were found to be non-random both at the nucleotide and dinucleotide sequence levels, an observation consistent with an endogenous sequence-directed mechanism of mutagenesis. Direct repeats of between 2bp and 8bp 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 length 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 approximately 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 alpha during DNA replication and (v) have been shown by in vitro studies of human polymerase alpha to be especially prone to frameshift mutation.(ABSTRACT TRUNCATED AT 400 WORDS)
Examples of the insertion of less than 10 bp of DNA sequence into human gene-coding regions causing genetic disease were collated in order to study the underlying causative mechanisms. The nature of these insertions was found to be consistent with several mechanisms of mutagenesis including: (1) slipped mispairing mediated by direct repeats or runs of identical bases and (2) the templated misincorporation of bases by secondary-structure intermediates whose formation is facilitated by palindromic (inverted repeat) sequences, quasi-palindromic sequences or symmetric elements. Both the size and position of insertions were found to be non-random and highly dependent upon the surrounding DNA sequence. Inferred mechanisms of insertional mutagenesis thus appear to be very similar to those involved in the causation of gene deletions.
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.
pJU78 is a 2.9-kb cloned human DNA segment derived from Xq24-q26. When used as a hybridization probe, it detects some 25 related sequences dispersed over the genome, including several autosomes and the X and Y chromosomes. Several pJU78-related sequences were chromosomally allocated and five different restriction fragment length polymorphisms detected and partially characterized in population and family studies. This sequence family was not found in non-primate species. The sequence appears to lack CpG-island character and is not detectably expressed in a variety of human tissues.
A case of homozygous factor X deficiency arising from the inheritance of two non-identical gene deletions from heterozygous parents is described. One, a partial gene deletion, was localized to exons VII and VIII by a combination of Southern blotting and polymerase chain reaction (PCR) amplification of exon sequences. The other deletion, of maternal origin, probably involves the entire factor X gene. Restriction fragments associated with the exon VII + VIII deletion were present in three siblings including the homozygous proband. These fragments were however absent from the somatic cells of the father, a finding consistent with germline mosaicism.
Recombinant DNA methodology has greatly increased our knowledge of the molecular pathology of the human genome at the same time as providing the means to diagnose inherited disease at the DNA level. Direct detection and analysis of a range of genetic defects are now possible using cloned gene or oligonucleotide probes or by direct sequencing of the disease gene(s). In addition, the use of restriction fragment length polymorphisms (RFLPs) within and around these genes as indirect genetic markers has not potentiated the tracking of disease alleles in affected pedigrees in cases where direct analysis was not feasible. RFLPs associated with linked anonymous segments may also be used not only to diagnose hitherto undetectable disease states, but also for chromosomal localization of the loci responsible. We present here an updated list of reports describing both the direct and the indirect analysis/diagnosis of human inherited disease; it is intended to serve as a guide to current molecular genetic approaches in diagnostic medicine.