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Biomedical subjects

P M Green

Publications and source records attributed to P M Green.

At least 19 recordsLinked to original sources
PubMed2001

Structural elements of the osteopontin SVVYGLR motif important for the interaction with alpha(4) integrins.

The osteopontin SVVYGLR motif binds the integrins alpha(4)beta(1) and alpha(9)beta(1). We show that alpha(4)beta(7) also interacts with this motif and that an SVVYGLR-OH peptide antagonises the alpha(4)beta(7) MAdCAM interaction. The important elements of this motif required to bind alpha(4)beta(1) and alpha(4)beta(7) were probed using a series of mutated peptides based around SVVYGLR. Leu167 is important for the interaction with alpha(4) integrins, as is the C-terminal carboxylic acid of Arg168 exposed by thrombin cleavage. The importance of the acidic group means that SVVYGLR has structural elements in common with other alpha(4) integrin-binding motifs and suggests why thrombin cleavage activates this motif.

Amino Acid Motifs↗
PubMed2000

The X chromosome and the rate of deleterious mutations in humans.

Monosomy for the X chromosome in humans creates a genetic Achilles' heel for nature to deal with. We report that the human X chromosome appears to have one-third the density of the coding sequence of the autosomes and, because of partial shielding from the high mutation rate of the male sex, that it should also have a lower mutation rate than the autosomes (i.e.,.73). Hence, the X chromosome should contribute one quarter (.33x.73=.24) of the deleterious mutations expected from its DNA content. In this way, selection has possibly moderated risks from mutation in X-linked genes that are thought to have been fixed in their syntenic state since the onset of the mammalian lineage. The unexpected difference in the density of coding sequences indicates that our recent, hemophilia B-based estimate of the rate of deleterious mutations per zygote should be increased from 1.3 to 4 (1.3x3).

Base Composition↗
PubMed2000

Biallelic discrimination assays for the three common Ashkenazi Jewish mutations and a common non-Jewish mutation, in Tay-Sachs disease, using fluorogenic TaqMan probes.

We have developed rapid semiautomated fluorogenic TaqMan assays for the three common Jewish mutations that occur in Tay-Sachs disease, the TATC 4-bp insertion in exon 11 (1,278insTATC), the IVS 12 + 1G --> C, splice site mutation in intron 12 (1421 + 1 G --> C), and the G --> A change at the 3' end of exon 7 (G269S), as well as for a non-Jewish mutation, IVS9 + I G --> A, believed to be prevalent in patients of Celtic descent. The TaqMan assays are designed to run on the ABI SDS 7700 sequence detection system, using allele-specific probes that carry a reporter dye at the 5' end and a quencher dye at the 3' end. Using a 96-well format, all four assays can be performed simultaneously on the same plate, with real-time fluorescence detection or just an end-point plate read. DNA samples from 78 patients identified as carriers by biochemical screening and genotyped by conventional techniques were used to assess the accuracy and efficiency of the probes in allelic discrimination assays. There were no discrepancies noted between previously assigned genotypes and the results obtained by application of this methodology.

Alleles↗
PubMed1999

Detection of mutations in COL4A5 in patients with Alport syndrome.

Alport syndrome (AS) can be caused by mutations in COL4A5, one of the six type IV collagen genes. For the purposes of confirming diagnoses, carrier screening and correlating genotype to phenotype, we have screened all 51 exons of this gene by SSCP analysis in 153 families with suspected AS. Mutations were identified in 77 families (of which 20 have previously been reported) and are reported with all available clinical information. All types of mutation were found (missense, nonsense, splicing, small and large deletions and insertions), with the commonest type being those affecting glycine residues in the collagen triple helix. Our 50% detection rate is similar to that of other groups and may imply the presence of mutations outside of the COL4A5 coding region or the existence of a second X-linked AS gene.

Adolescent↗
PubMed1999

Creation of a novel donor splice site in intron 1 of the factor VIII gene leads to activation of a 191 bp cryptic exon in two haemophilia A patients.

We have constructed a confidential U.K. database of haemophilia A mutations and pedigrees by characterizing the gene defect of one index patient in each U.K. family. Mutations were identified by screening all coding regions of the factor VIII (FVIII) mRNA, using solid-phase fluorescent chemical cleavage of mismatch and examining additional non-coding regions of the gene. Here we report two haemophilia A patients (UK 114 FVIII:C 2% and UK 243 FVIII:C < 1%) with an abnormal FVIII mRNA due to an A to G point mutation, 1.4 kb downstream from exon 1 in the FVIII gene. This mutation creates a new donor splice site in intron 1 and leads to insertion of a 191 bp novel exon in the mRNA. Haplotype analysis suggests that the mutation may have originated in a common ancestor of the two patients, who further illustrate how mRNA analysis allows higher efficiency of haemophilia A mutation detection, because their mutation would not have been identified by direct analysis of the factor VIII gene.

Base Sequence↗
PubMed1999

DNA variation in a 5-Mb region of the X chromosome and estimates of sex-specific/type-specific mutation rates.

We describe a new approach for the study of human genome variation, based on our solid-phase fluorescence chemical mismatch-cleavage method. Multiplex screening rates >/=80 kb/36-lane gels are achieved, and accuracy of mismatch location is within +/-2 bp. The density of differences between DNA from any two humans is sufficiently low, and the estimate of their position is accurate enough, to avoid sequencing of most polymorphic sites when defining their allelic state. Furthermore, highly variable sequences, such as microsatellites, are distinguished easily, so that separate consideration can be given to loci that do and do not fit the definition of infinite mutation sites. We examined a 5-Mb region of Xq22 to define the haplotypes of 23 men (9 Europeans, 9 Ashkenazim, and 5 Pygmies) by reference to DNA from one Italian man. Fifty-eight 1.5-kb segments revealed 102 segregating sites. Seven of these are shared by all three groups, two by Pygmies and Europeans, two by Pygmies and Ashkenazim, and 19 by Ashkenazim and Europeans. Europeans are the least polymorphic, and Pygmies are the most polymorphic. Conserved allelic associations were recognizable within 40-kb DNA segments, and so was recombination in the longer intervals separating such segments. The men showed only three segregating sites in a 16.5-kb unique region of the Y chromosome. Divergence between X- and Y-chromosome sequences of humans and chimpanzees indicated higher male mutation rates for different types of mutations. These rates for the X chromosomes were very similar to those estimated for the X-linked factor IX gene in the U.K. population.

Animals↗
PubMed1999

Mutation rates in humans. I. Overall and sex-specific rates obtained from a population study of hemophilia B.

A population-based study of hemophilia B mutations was conducted in the United Kingdom in order to construct a national confidential database of mutations and pedigrees to be used for the provision of carrier and prenatal diagnoses based on mutation detection. This allowed the direct estimate of overall (micro), male (v), and female (u) mutation rates for hemophilia B. The values obtained per gamete per generation and the 95% confidence intervals are micro;=7.73 (6. 29-9.12&parr0;x10-6; v=18.8 (14.5-22.9&parr0;x10-6; and u=2.18 (1. 44-3.16&parr0;x10-6. The ratio of male-to-female mutation rates is 8. 64, with a 95% confidence interval of 5.46-14.5. The higher male rate was not caused by a much higher rate of transition at CpG sites in the male. Attempts to detect evidence of gonadal mosaicism for hemophilia B mutation in suitable families did not detect any instances of ovarian mosaicism in any of 47 available opportunities. This suggests that the risk of a noncarrier mother manifesting as a gonadal mosaic by transmitting the mutation to a second child should be <0.062.

CpG Islands↗
PubMed1999

Mutation rates in humans. II. Sporadic mutation-specific rates and rate of detrimental human mutations inferred from hemophilia B.

We estimated the rates per base per generation of specific types of mutations, using our direct estimate of the overall mutation rate for hemophilia B and information on the mutations present in the United Kingdom's population as well as those reported year by year in the hemophilia B world database. These rates are as follows: transitions at CpG sites 9.7x10-8, other transitions 7.3x10-9, transversions at CpG sites 5.4x10-9, other transversions 6.9x10-9, and small deletions/insertions causing frameshifts 3.2x10-10. By taking into account the ratio of male to female mutation rates, the above figures were converted into rates appropriate for autosomal DNA-namely, 1.3x10-7, 9.9x10-9, 7.3x10-9, 9.4x10-9, 6.5x10-10, where the latter is the rate for all small deletion/insertion events. Mutation rates were also independently estimated from the sequence divergence observed in randomly chosen sequences from the human and chimpanzee X and Y chromosomes. These estimates were highly compatible with those obtained from hemophilia B and showed higher mutation rates in the male, but they showed no evidence for a significant excess of transitions at CpG sites in the spectrum of Y-sequence divergence relative to that of X-chromosome divergence. Our data suggest an overall mutation rate of 2.14x10-8 per base per generation, or 128 mutations per human zygote. Since the effective target for hemophilia B mutations is only 1.05% of the factor IX gene, the rate of detrimental mutations, per human zygote, suggested by the hemophilia data is approximately 1.3.

Animals↗
PubMed1999

A study of anal intraepithelial neoplasia in HIV positive homosexual men.

OBJECTIVES: To determine the prevalence of high grade anal intraepithelial neoplasia (HGAIN), the value of anal cytology in screening for HGAIN, and the characterisation of epidemiological factors and human papillomavirus (HPV) types. METHODS: Prospective cohort study of HIV positive homosexual men. Subjects were interviewed, underwent STD, anal cytological, and HPV screening at enrolment and at subsequent follow up visits with anoscopy and biopsy at the final visit. 57 enrolled, average CD4 count 273 x 10(6)/l (10-588); 41 completed the cytological surveillance over the follow up period (181 visits, average follow up 17 months), 38 of these had anoscopy and anal biopsy. RESULTS: Oncogenic HPV types were detected in 84% and high grade dyskaryosis in 10.5% (6/57) at enrollment. There was a 70% incidence of high grade dyskaryosis during follow up in patients with negative/warty or low grade dyskaryosis at enrollment. Anoscopy correlated with histology in high grade AIN lesions (sensitivity 91%, specificity 54%) and cytology was 78% sensitive (18/23) for HGAIN on biopsy. CONCLUSIONS: AIN and infection with multiple oncogenic HPV types are very common among immunosuppressed HIV positive homosexual men. Apparent progression from low to high grade cytological changes occurred over a short follow up period, with no cases of carcinoma. All 23 cases of HGAIN were predicted by cytology and/or anoscopy. Future studies focusing on the risk of progression to carcinoma are needed before applying anal cytology as a screening tool for AIN in this population.

Adult↗
PubMed1999

External cephalic version.

The management of breech presentation at term is variable. This article presents the options, and reviews the current evidence for, and success rate of, external cephalic version. The work of the breech clinic at the Liverpool Women's Hospital is also discussed.

Algorithms↗
PubMed1999

Start of UK confidential haemophilia A database: analysis of 142 patients by solid phase fluorescent chemical cleavage of mismatch. Haemophilia Centres.

A national strategy for optimising genetic services in haemophilia A has been initiated in the UK. Solid phase fluorescent chemical cleavage of mismatch is used to screen the entire coding region of factor VIII in six segments: four amplified from the trace of mRNA in blood lymphocytes and two from genomic DNA for the 3.4 kb exon 14 and flanking intron sequences. These segments are analysed in two threefold multiplexes so that the genes of 18 patients can be screened in a single ABI 377 gel. The promoter and polyadenylation signal region are amplified and sequenced directly. We have analysed 142 unrelated patients and identified 141 factor VIII mutations and one Normandy type von Willebrand homozygote. The former mutations include 89 missense, 10 nonsense, 5 frameshift, one 24 bp deletion and one splice signal defect. These comprise 71 different changes, of which 39 have not been previously observed.

Base Pair Mismatch↗
PubMed1999

Genomic organization and chromosomal localization of the human CUL2 gene and the role of von Hippel-Lindau tumor suppressor-binding protein (CUL2 and VBP1) mutation and loss in renal-cell carcinoma development.

Germline mutations in the von Hippel-Lindau (VHL) disease tumor suppressor gene (TSG) convey a high risk of clear-cell renal-cell carcinoma (CC-RCC) and most sporadic CC-RCCs demonstrate somatic inactivation of the VHL TSG. However, the existence of further CC-RCC gatekeeper genes is implied by CC-RCC kindreds not linked to the VHL gene and the absence of somatic VHL inactivation in approximately 30% of sporadic CC-RCC. Genes that encode proteins which interact with the VHL gene product (VHL) provide candidate gatekeeper RCC genes. VHL forms a multimeric complex with two subunits (B and C) of the SIII (elongin) transcriptional elongation complex and CUL2, a member of the cullin family. Most pathogenic VHL mutations inhibit formation of the VHL/elonginB+C/CUL2 complex. A further VHL-binding protein of unknown function, VBP1, fails to bind to truncated forms of VHL. We have investigated the possible roles of CUL2 and VBP1 in renal tumorigenesis by analyzing sporadic RCC of known VHL mutation or hypermethylation status, including CC-RCC without VHL inactivation (n = 40); CC-RCC with VHL inactivation (n = 35); and non-CC-RCC (n = 14). No VBP1 mutations were identified in 89 sporadic RCCs, suggesting that VBP1 is not an RCC gatekeeper gene. To investigate CUL2, we mapped the CUL2 gene to chromosome band 10p11.1-p11.2, a region reported to show loss of heterozygosity (LOH) in several human cancers (including non-CC-RCC); determined the genomic organization; and performed mutation analysis of the 21 exons identified. Using novel intragenic polymorphisms, we detected LOH in 6/25 informative RCCs; however, no pathogenic CUL2 mutations were identified in the 89 RCCs analyzed. These findings suggest that unless CUL2 is inactivated by epigenetic events, it is not a major RCC TSG. However, CUL2 remains a candidate TSG for other tumor types demonstrating 10p LOH. Genes Chromosomes Cancer 26:20-28, 1999.

Carcinoma, Renal Cell↗
PubMed1998

Haemophilia B: database of point mutations and short additions and deletions--eighth edition.

The eighth edition of the haemophilia B database (http://www.umds.ac. uk/molgen/haemBdatabase.htm ) lists in an easily accessible form all known factor IX mutations due to small changes (base substitutions and short additions and/or deletions of <30 bp) identified in haemophilia B patients. The 1713 patient entries are ordered by the nucleotide number of their mutation. Where known, details are given on: factor IX activity, factor IX antigen in circulation, presence of inhibitor and origin of mutation. References to published mutations are given and the laboratories generating the data are indicated.

Computer Communication Networks↗
PubMed1998

Sequence, exon-intron organization, transcription and mutational analysis of prnA, the gene encoding the transcriptional activator of the prn gene cluster in Aspergillus nidulans.

The prnA gene codes for a transcriptional activator that mediates proline induction of four other genes involved in proline utilization as a nitrogen and/or carbon source in Aspergillus nidulans. In this paper, we present the genomic and cDNA sequence and the transcript map of prnA. The PrnA protein belongs to the Zn binuclear cluster family of transcriptional activators. The gene shows a striking intron-exon organization, with the putative nuclear localization sequence and the Zn cluster domain in discrete exons. Although the protein sequence presents some interesting similarities with the isofunctional protein of Saccharomyces cerevisiae Put3p, a higher degree of similarity is found with a functionally unrelated protein Thi1 of Schizosaccharomyces pombe. A number of mutations mapping in the prnA gene were sequenced. This comprises a deletion that results in an almost complete loss of the prnA-specific mRNA, a mutation in the putative nuclear localization signal, a proline to leucine mutation in the second loop of the zinc cluster and a cold-sensitive mutation in the so-called 'central region'. Other complete or partial loss of function mutations map in regions of unknown function. We establish that the transcription of the gene is neither self-regulated nor significantly affected by carbon and/or nitrogen metabolite repression.

Aspergillus nidulans↗
PubMed1997

Characterization of the gene (VBP1) and transcript for the von Hippel-Lindau binding protein and isolation of the highly conserved murine homologue.

A single-copy, widely expressed gene of at least 30 kb and six exons was discovered via a hybrid mRNA resulting from an inversion causing hemophilia A. A segment of the 1.7-kb message of this gene has been shown by others to encode a protein (named VBP1) interacting with the product of the von Hippel-Lindau gene and thus is expected to participate in pathways involving this tumor suppressor gene. The mouse VBP1 message we isolated encodes a polypeptide of 160 residues absolutely identical to that of human. Even the 3' untranslated tails of the mRNAs show 68% conservation, and both use the unusual ATTAAA polyadenylation signal. The mouse gene has a single transcription start while the human homologue has two major starts and a minor start. This could result in amino-end extensions of the human protein. A polymorphism with 42% heterozygosity in the United Kingdom population was detected in the 3' tail of the message. VBP1 is unlike other known proteins but a consensus for tyrosine phosphorylation possibly suggests regulation by kinases.

Animals↗
PubMed1997

Haemophilia B: database of point mutations and short additions and deletions, 7th edition.

The seventh edition of the haemophilia B database lists in easily accessible form all known factor IX mutations due to small changes (base substitutions and short additions and/or deletions of <30 bp) identified in haemophilia B patients. The 1535 patient entries are ordered by the nucleotide number of their mutation. Where known, details are given on: factor IX activity, factor IX antigen in circulation, presence of inhibitor and origin of mutation. References to published mutations are given and the laboratories generating the data are indicated.

Databases, Factual↗