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Biomedical subjects

I R Peake

Publications and source records attributed to I R Peake.

At least 37 records · Page 2Linked to original sources
PubMed2000

Relationship between factor VIII mutation type and inhibitor development in a cohort of previously untreated patients treated with recombinant factor VIII (Recombinate). Recombinate PUP Study Group.

A cohort of 79 previously untreated patients (PUPs) with moderate-severe haemophilia A (baseline Factor VIII < or =2%) were enrolled in a study to evaluate the safety, efficacy and immunogenicity of recombinant factor VIII (r-FVIII, Recombinate). Blood samples were obtained retrospectively from a total 55 PUPs who were investigated for the spectrum of FVIII gene mutations responsible for their haemophilia. FVIII gene inversion mutations were found in 27 (49%) patients. Two patients had partial gene deletions. The remaining 26 patients were then screened for mutations in the FVIII gene coding region using conformation sensitive gel electrophoresis. Point mutations were identified in 22 (85%) of the patients and 14 of these mutations were novel. Study subjects were monitored for the development of FVIII inhibitors throughout the study. A total of 23 of the 73 evaluable subjects (including one subject with a low inhibitor titer at baseline) demonstrated an inhibitor on one or more occasions; 11 (15%) were persistent. Inhibitors were detected in patients with partial gene deletions and inversions and in three of eight patients with missense mutations. No inhibitors were found in 11 patients with small insertions or deletions resulting in an alteration of the protein translation reading frame (frameshift mutations). The results corroborate the observation that mutation type is an important determinant of the propensity to develop inhibitory anti-FVIII antibody.

Antibody Formation↗
PubMed1999

A rapid method for haemophilia B mutation detection using conformation sensitive gel electrophoresis.

Conformation sensitive gel electrophoresis (CSGE) was confirmed as an effective procedure for screening the factor IX (FIX) gene by detecting 10/10 previously known FIX gene mutations. The FIX genes of a further 11 haemophilia B patients with unknown mutations were then screened and an abnormal CSGE profile was identified in all cases. Subsequent DNA sequencing demonstrated one of these to be a novel mutation (31133insT, Arg338Fs), the remaining 10 having been previously reported on the haemophilia B database. Mutation screening of the FIX gene using CSGE was demonstrated to be a rapid and efficient means of carrier analysis in families with haemophilia B.

Electrophoresis, Agar Gel↗
PubMed1999

A common splice site mutation is shared by two families with different type 2N von Willebrand disease mutations.

Using an ELISA-based method to detect type 2N von Willebrand disease (VWD), we found two individuals with absent FVIII binding. Direct sequencing of the FVIII binding region of the von Willebrand factor (VWF) gene showed that one individual had an R854Q substitution whilst the other had a T791M substitution. The very low FVIII binding and the VWF:Ag levels in both individuals suggested a second defect on the other VWF allele. Conformation sensitive gel electrophoresis of polymerase chain reaction amplified DNA was used to detect an additional change in the VWF gene of each patient. Direct sequencing confirmed a previously unreported G to A transition in the donor splice site in intron 25 of both individuals which should result in a null allele. This was confirmed by mRNA analysis. These two individuals therefore have compound heterozygous VWD in which the only expressed allele has a type 2N mutation. In our population, such compound heterozygosity appears to be a significant cause of type 2N VWD.

Alleles↗
PubMed1998

Antithrombins Wibble and Wobble (T85M/K): archetypal conformational diseases with in vivo latent-transition, thrombosis, and heparin activation.

The inherent variability of conformational diseases is demonstrated by two families with different mutations of the same conserved aminoacid in antithrombin. Threonine 85 underlies the opening of the main beta-sheet of the molecule and its replacement, by the polar lysine, in antithrombin Wobble, resulted in a plasma deficiency of antithrombin with an uncharacteristically severe onset of thrombosis at 10 years of age, whereas the replacement of the same residue by a nonpolar methionine, antithrombin Wibble, gave near-normal levels of plasma antithrombin and more typical adult thromboembolic disease. Isolated antithrombin Wibble had a decreased thermal stability (Tm 56.2, normal 57.6 degreesC) but was fully stabilized by the heparin pentasaccharide (Tm 71.8, normal 71.0 degreesC), indicating that the prime abnormality is a laxity in the transition of the main sheet of the molecule from the 5- to 6-stranded form, as was confirmed by the ready conversion of antithrombin Wibble to the 6-stranded latent form on incubation. That this transition can occur in vivo was shown by the finding of nearly 10% of the proband's plasma antithrombin in the latent form and also, surprisingly, of small but definitive amounts of latent antithrombin in normal plasma. The latent transition will be predictably accelerated not only by gross mutations, as with antithrombin Wobble, to give severe episodic thrombosis, but also by milder mutations, as with antithrombin Wibble, to trigger thrombosis in the presence of other predisposing factors, including the conformational stress imposed by the raised body temperatures of fevers. Both antithrombin variants had an exceptional (25-fold) increase in heparin affinity and this, together with an increased inhibitory activity against factor Xa, provides evidence of the direct linkage of A-sheet opening to the conformational basis of heparin binding and activation.

Amino Acid Substitution↗
PubMed1998

Precise carrier diagnosis in families with haemophilia A: use of conformation sensitive gel electrophoresis for mutation screening and polymorphism analysis.

Causative mutations in the factor VIII gene of seven unrelated patients with severe haemophilia A were identified using the mutation screening procedure conformation sensitive gel electrophoresis (1) and characterised by direct sequencing. Female family members of all patients had requested either carrier status determination or prenatal diagnosis. However, lack of the factor VIII gene inversion, a prior family history or informative polymorphisms prevented diagnosis in these families. Identification of a mutation in each family enabled female carrier status to be determined in all cases. Six mutations were previously unreported. One Afro-Caribbean patient had two sequence changes; A670 2G and A6769G. The latter, resulting in Met2238Val and previously reported as a FVIII mutation, was shown to be polymorphic with a 42% heterozygosity rate in an Afro-Caribbean population. Conformation sensitive gel electrophoresis was found to be technically simple and efficient at locating previously unknown FVIII gene mutations.

Blotting, Southern↗
PubMed1998

Factor VIII inhibitors in mild and moderate-severity haemophilia A. UK Haemophilia Centre Directors Organisation.

Twenty six patients with mild or moderate haemophilia A and inhibitors are described. The inhibitor was detected at a median age of 33 years, after a median of 5.5 bleeding episodes. This usually following intensive replacement therapy. The median presenting inhibitor titre was antihuman 11.6 BU/ml, antiporcine 1.45 BU/ml. Plasma basal factor VIII level declined from a median of 0.08 IU/ml to 0.01 IU/ml following the inhibitor development. This caused spontaneous bleeding in 22 and a bleeding pattern similar to acquired haemophilia in 17. Bleeding was often severe and caused two deaths. The inhibitor disappeared spontaneously, or following immune tolerance induction, in 16 cases after a median of 9 months (range 0.5-46), with a return to the original baseline VIIIC level and bleeding pattern accompanied inhibitor loss. The inhibitor persisted in the remainder of the cases over a median period of 99 months (range 17-433 months) of follow-up. Inhibitors are an uncommon complication of mild haemophilia which frequently persist and may be associated with severe, life-threatening, haemorrhage. Forty-one percent of treated haemophilic family members had a history of factor VIII inhibitors, suggesting a familial predisposition to develop inhibitors in these kindreds. Sixteen patients from 11 families were genotyped. Seven different missense mutations affecting the light chain were detected and two in the A2 domain. Five patients from three families had a mutation causing a substitution of Trp2229 by Cys in the C2 domain which appears to predispose to inhibitor formation since 7 of the 18 affected individuals have a history of inhibitor development.

Adolescent↗
PubMed1998

A novel mutation in intron K of the PROS1 gene causes aberrant RNA splicing and is a common cause of protein S deficiency in a UK thrombophilia cohort.

In the course of investigating the molecular basis of protein S deficiency in 31 index cases with thrombophilia, we identified seven kindred where the underlying defect was a novel A to G transition 9 bp upstream of exon 12 in intron K of the PROS1 gene. In all but one case, the mutation caused type I deficiency. One individual had type III deficiency. While ectopic transcript analysis using the BstXI dimorphism in exon 15 failed to detect a transcript from the mutated allele, analysis of transcripts spanning exons 11 and 12 revealed a minor mRNA species. Sequencing confirmed the mutation created a new RNA acceptor site introducing 8 nucleotides of intronic sequence into the mature mRNA. Haplotype analysis of the defective PROS1 alleles in six families revealed the same haplotype in all affected individuals suggesting the presence of a common ancestor. Six of the fourteen relatives with the mutation experienced at least one venous thrombotic event strongly supporting the association of the mutation with venous thrombosis.

Alleles↗
PubMed1997

HLA class II profile: a weak determinant of factor VIII inhibitor development in severe haemophilia A. UKHCDO Inhibitor Working Party.

The risk of developing factor VIII inhibitor antibodies in haemophilia A may relate both to factor VIII genotype and genes within the HLA complex known to influence immune response. We investigated a cohort of 176 patients with severe haemophilia A and with either high-level inhibitors (> 10BU/ml) or with no history of an inhibitor, stratified according to the presence or absence of the factor VIII gene intron 22 inversion. HLA DRB1, DQA1 and DQB1 polymorphisms were determined by PCR. HLA frequencies form 137 United Kingdom controls were used for comparison. HLA phenotype frequency differences, expressed as odds ratios with 95% confidence intervals were as follows: HLA-DRB*1501, DQB1*0602 and DQA1*0102 were all increased in frequency in patients with inhibitors, only DQA1*0102 reaching statistical significance (OR 2.7, 1.2-5.9). These alleles form part of an established HLA haplotype. The frequencies of HLA-DRB1*1501, DQB1*0602 and DQA1*0102 were particularly raised in patients with inhibitors and a factor VIII gene intron 22 inversion, although again only DQA1*0102 achieved significance (OR 3.1, 1.0-10.1). The frequency of DRB1*01, DQB1*0501, DQA1*0101 were also increased in inhibitor patients lacking the intron 22 inversion although this failed to achieve statistical significance. This data suggests that HLA class II profile constitutes a weak risk factor for developing inhibitor antibodies to factor VIII. This may be more pronounced in patients with an intron 22 inversion.

Chromosome Inversion↗
PubMed1997

Co-inheritance of the 20210A allele of the prothrombin gene increases the risk of thrombosis in subjects with familial thrombophilia.

The presence of the 20210A allele of the prothrombin (PT) gene has recently been shown to be a risk factor for venous thromboembolism. This is probably mediated through increased plasma prothrombin levels. The aim of this study was to compare the prevalence of the prothrombin 20210A allele in control subjects and in subjects with recognised thrombophilia and to establish whether the additional inheritance of the PT 20210A allele is associated with an increased risk of venous thromboembolism. 101 subjects with a history of venous thromboembolism and diagnosed as having either factor V Leiden (R506Q) or heritable deficiencies of protein C, protein S or antithrombin were studied. The prevalence of the PT 20210A allele in this group was compared with the results obtained for 150 control subjects. In addition, the relationships were examined between genetic status and the number of documented thromboembolic episodes, and between plasma prothrombin levels and possession of the PT 20210A allele. 8 (7.9%) of the 101 patients were also heterozygous for the PT 20210A allele. This compares with 0.7% in the control subjects (p = 0.005). After exclusion of patients on warfarin, the mean plasma prothrombin of 113 subjects without 20210A was 1.09 U/ml, as compared with 1.32 U/ml in 8 with the allele (p = 0.0002). Among the 101 patients with either factor V Leiden, protein S deficiency, protein C deficiency or antithrombin deficiency, the age adjusted mean (SD) number of venous thromboembolic episodes at diagnosis was 3.7 (1.5) in those with the PT 20210A allele, as compared with 1.9 (1.1) in those without (p = 0.0001). We have demonstrated that the prevalence of the PT 20210A allele is significantly greater in subjects with venous thrombosis and characterised heritable thrombophilia than in normal control subjects and that the additional inheritance of PT 20210A is associated with an increased risk of venous thromboembolism. We have also confirmed that plasma prothrombin levels are significantly greater in subjects possessing the PT 20210A compared with those who do not.

Adolescent↗
PubMed1996

Molecular basis of protein S deficiency in three families also showing independent inheritance of factor V leiden.

The molecular basis of type I or III Protein S deficiency has been investigated in three kindred also showing independent inheritance of factor V (FV) Leiden. A T to C transition in codon 570 (Met-->Thr) was identified in the propositi and shown to segregate with protein S deficiency in all but one of the affected members of two kindred. This individual was heterozygous for a second transition (C to T) causing substitution of serine 624 by leucine. A second member of the same family, with markedly reduced free protein S levels when compared with affected relatives, was heterozygous for both mutations. Haplotype analysis of individuals with the mutated ATG570ACG allele in the two kindred suggested they may have been related by a common ancestor. A G to A transition resulting in substitution of cysteine 145 by tyrosine was detected in the third kindred. All mutations are believed to interfere with protein S binding to C4b-binding protein resulting in reduced free protein S levels. Of the five individuals studied who had experienced thrombotic events, three had combined protein S deficiency and FV Leiden reemphasising the importance of FV Leiden as an additional risk factor for thrombosis in protein S deficiency.

Alleles↗
PubMed1996

A rapid and cost effective method for analysis of dinucleotide repeat polymorphisms in the factor VIII gene.

A simple, rapid and cheap method for the analysis of variable dinucleotide tandem polymorphisms in introns 13 and 22 of the factor VIII gene has been established. The protocol uses blood spots stored on filter paper (Guthrie spots) and other DNA containing materials as sources of DNA. Following DNA amplification using a thermostable DNA polymerase, products are size fractionated on native polyacrylamide gels and visualized by silver staining. This simplified protocol obviates the use of DNA extraction, reducing time and costs and in addition, reduces the requirement for gel documentation equipment (i.e., photography), as silver staining can be visualized readily without additional equipment and the gels themselves can be stored. These alterations to the analysis enhance the universal applicability of these polymorphisms, as was demonstrated by a comparative study in Caucasian and Thai females.

Cost-Benefit Analysis↗
PubMed1996

Characterisation of type 2N von Willebrand disease using phenotypic and molecular techniques.

von Willebrand factor (vWF) is a multimeric glycoprotein found in plasma non covalently linked to factor VIII (FVIII). Type 2N von Willebrand disease (vWD) is caused by a mutation in the vWF gene that results in vWF with a normal multimeric pattern, but with reduced binding to FVIII. We have utilised methods for the phenotypic and genotypic detection of type 2N vWD. The binding of FVIII to vWF in 69 patients, 36 with type 1 vWD, 32 with mild haemophilia A and one possible haemophilia A carrier with low FVIII levels was studied. Of these, six were found to have reduced binding (five type 1 vWD, one possible haemophilia A carrier). DNA was extracted from these patients and exons 18-23 of the vWF gene encoding the FVIII binding region of vWF were analysed. After direct sequencing and chemical cleavage mismatch detection, a Thr28Met mutation was detected in two unrelated individuals, one of whom appears to be a compound heterozygote for the mutation and a null allele. No mutations were found in the region of the vWF gene encoding the FVIII binding region of vWF in the other four patients.

Alleles↗
PubMed1995

Rapid genotype analysis in type 2B von Willebrand's disease using a universal heteroduplex generator.

A new diagnostic technique based on DNA heteroduplex analysis has been used to identify specific point mutations in the von Willebrand's factor (vWF) gene of patients with von Willebrand's disease type 2B. Molecular analysis in these patients has shown previously that their mutations are clustered in a short region of sequence in exon 28 of the vWF gene. The principle of the method involves heteroduplex formation between amplified genomic sequence containing the defect and an exon 28 vWF gene universal heteroduplex generator (UHG). The UHG is a synthetic vWF gene exon 28 homologue which contains a number of sequence mismatches designed to generate allele specific heteroduplexes for each type 2B mutation. Individual mutant genotypes are identified by characteristic banding patterns following polyacrylamide minigel electrophoresis. The technique is rapid, simple, inexpensive, and is ideally suited for adoption by non-specialist haematology laboratories for screening purposes.

Base Sequence↗
PubMed1995

DNA polymorphisms for carrier detection of hemophilia in Thailand.

The assessment of carrier state based on the pedigree and laboratory testing in 55 females from 34 Thai hemophilia families (24 affected by hemophilia A, 10 by hemophilia B) was studied. The laboratory testing included phenotypic analysis (FVIII:C/vWF: Ag ratio, FIX:C) and two types of DNA polymorphisms, restriction fragment length polymorphisms (RFLP) and variable number tandem repeats (VNTR) in/and close to the factor VIII genes (Bcl I, Xba I RFLP, St 14 VNTR) and factor IX genes (Mse I, Dde I RFLP). Fifteen out of seventeen (88%) obligate hemophilia A carriers and one out of five (20%) obligate hemophilia B carriers were diagnosed by phenotypic analysis. All hemophilia A carriers were informative for at least one polymorphism (Bcl I, Xba I or St 14) while 42% of hemophilia B carriers were informative for Mse I RFLP only. DNA polymorphism analysis has advantage over phenotypic analysis since it generally gives an absolute diagnosis when informative. Most DNA polymorphism analyses are performed by PCR technique which is a simple, inexpensive and quick procedure. However, it is limited by non-informativeness and high incidence of new mutations.

Factor IX↗