Precise Carrier Diagnosis in Families with Haemophilia A: Use of Conformation Sensitive Gel Electrophoresis for Mutation Screening and Polymorphism Analysis

Williams, I. J.; Abuzenadah, Adel M.; Winship, Peter R.; Preston, F. E.; Dolan, G.; Wright, J.; Peake, I. R.; Goodeve, Anne

doi:10.1055/s-0037-1615052

Cited by 86 publications

(92 citation statements)

References 12 publications

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“…F8 mutations were analyzed using either confirmation sensitive gel electrophoresis followed by DNA sequencing of amplicons displaying a migration shift 12 or by direct sequencing of the entire coding region and intron-exon boundaries of F8 13 . Nucleotide numbering of the F8 cDNA was from the A of the ATG initiation codon and protein from the initiator methionine at the start of FVIII using reference sequences for cDNA NM_000132.2 and protein NP_000123.1.…”

Section: Genetic Analysismentioning

confidence: 99%

Specific and global coagulation assays in the diagnosis of discrepant mild hemophilia A

Bowyer¹,

Veen²,

Goodeve

et al. 2013

Haematologica

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The activity of the factor VIII coagulation protein can be measured by three methods: a one or two-stage clotting assay and a chromogenic assay. The factor VIII activity of most individuals with mild hemophilia A is the same regardless of which method is employed. However, approximately 30% of patients show marked discrepancies in factor VIII activity measured with the different methods. The objective of this study was to investigate the incidence of assay discrepancy in our center, assess the impact of alternative reagents on factor VIII activity assays and determine the usefulness of global assays of hemostasis in mild hemophilia A. Factor VIII activity was measured in 84 individuals with mild hemophilia A using different reagents. Assay discrepancy was defined as a two-fold or greater difference between the results of the one-stage and two-stage clotting assays. Rotational thromboelastometry and calibrated automated thrombography were performed. Assay discrepancy was observed in 31% of individuals; 12% with lower activity in the two-stage assay and 19% with lower activity in the one-stage assay. The phenotype could not always be predicted from the individual's genotype. Chromogenic assays were shown to be a suitable alternative to the two-stage clotting assay. Thromboelastometry was found to have poor sensitivity in hemophilia. Calibrated automated thrombography supported the results obtained by the two-stage and chromogenic assays. The current international guidelines do not define the type of assay to be used in the diagnosis of mild hemophilia A and some patients could be misclassified as normal. In our study, 4% of patients would not have been diagnosed on the basis of the one-stage factor VIII assay. Laboratories should use both one stage and chromogenic (or two-stage) assays in the diagnosis of patients with possible hemophilia A.

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Section: Genetic Analysismentioning

confidence: 99%

Specific and global coagulation assays in the diagnosis of discrepant mild hemophilia A

Bowyer¹,

Veen²,

Goodeve

et al. 2013

Haematologica

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“…To screen for additional F8 gene mutations, samples were tested for the intron 1 inversion 14 and analysed by conformation sensitive gel electrophoresis (CSGE) 15 by Dr Lillicrap, National Program for Hemophilia Mutation Testing, Department of Pathology and Molecular Medicine, Kingston, Ontario. Together with the F8 intron 22 inversion test, these techniques detect 85 -90% of F8 gene mutations (Dr Lillicrap, personal communications).…”

Section: Conformation Sensitive Gel Electrophoresismentioning

confidence: 99%

Heritable skewed X-chromosome inactivation leads to haemophilia A expression in heterozygous females

et al. 2007

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Factor VIII gene, F8, mutations cause haemophilia A (HA), an X-linked recessive disorder. Expression in heterozygous females has been ascribed to skewed X-chromosome inactivation (XCI). To investigate the cause of HA in three heterozygous females within an Atlantic Canadian kindred, the proband (severely affected girl, FVIII activity: 2%) and 17 relatives across three generations were studied. F8 genotype, FVIII activity, XCI ratio (XCIR) (paternal active X: maternal active X), karyotype, submegabase resolution tiling set array competitive genome hybridization (competitive genomic hybridization (SMRT)), and microsatellite analyses were utilized. A positive linear relationship between FVIII activity and percentageactivated normal X-chromosome was found in HA heterozygous females (R 2 ¼ 0.87). All affected, but no unaffected females, had an XCIR skewed toward activation of the mutant X-chromosome (proband 92:8, SD 2). Unexpectedly, high numbers of females have dramatically skewed XCIRs (480:20 or o20:80) (Po0.05). The distribution of XCIR frequencies within this family was significantly different than predicted by normal population data or models of random XCI (Po0.025), with more females having higher degrees of skewing. Known causes of skewing, such as chromosomal abnormalities, selection against deleterious alleles, and X-inactive-specific transcript mutations, are not consistent with our results. This study shows that FVIII activity in HA heterozygous females can be directly related to XCI skewing, and that low FVIII activity in females in this family is due to unfavourable XCI skewing. Further, the findings suggest that these XCI ratios are genetically influenced, consistent with a novel heritable human X controlling element (XCE) functioning similarly to the mouse Xce.

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“…The entire F8C coding region, including the flanking splicing sites, was amplified using the appropriate primers as described [14]. The F8C promoter sequence and the polyadenylation site were analyzed using the primers described elsewhere [15]. Equal volumes of PCR product from each patient and from a wild-type control male were mixed and treated to obtain heteroduplexes.…”

Section: Dna Extraction and Mutation Analysismentioning

confidence: 99%

Small FVIII gene rearrangements in 18 hemophilia A patients: Five novel mutations

et al. 2005

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Hemophilia A (HA) is a disorder caused by mutations of the FVIII gene, which is located on the tip of the long arm of the X chromosome. In a cohort of 18 unrelated Italian patients affected with HA of varying severity, we performed mutational screening of the gene by denaturing high-performance liquid chromatography (DHPLC) and direct sequencing of abnormal peaks. We identified five novel mutations and 9 previously reported DNA alterations. Two of the 9 previously reported alterations were each common to 3 unrelated patients. Six different mutations were characterized as missense alterations, while 8 were non-missense mutations. Among the new gene alterations, one created a stop codon, one consisted of an out-of frame deletion, and one was a splice-site mutation. The last two were missense alterations. In an attempt to better understand the causative effect of the mutations and the clinical variability of the patients, we investigated the consequences of each missense mutation and visualized the effect of the amino acid change on structural FVIII models. Am.

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Precise Carrier Diagnosis in Families with Haemophilia A: Use of Conformation Sensitive Gel Electrophoresis for Mutation Screening and Polymorphism Analysis

Cited by 86 publications

References 12 publications

Specific and global coagulation assays in the diagnosis of discrepant mild hemophilia A

Specific and global coagulation assays in the diagnosis of discrepant mild hemophilia A

Heritable skewed X-chromosome inactivation leads to haemophilia A expression in heterozygous females

Small FVIII gene rearrangements in 18 hemophilia A patients: Five novel mutations

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