Improved mutation detection for haemophilia A in South Africa

Show simple item record Mitchell, Claire Lynne 2009-11-03T11:19:09Z 2009-11-03T11:19:09Z 2009-11-03T11:19:09Z
dc.description M.Sc. (Med.), Faculty of Health Sciences, University of the Witwatersrand, 2009. en_US
dc.description.abstract Haemophilia A is a common X-linked recessive bleeding disorder, affecting about 1 in 5000 males worldwide. It is caused by a deficiency of functional coagulation Factor VIII (FVIII), resulting in prolonged or abnormal bleeding episodes. The severity of the disease is related to the level of functional FVIII in the plasma. The FVIII gene is a large gene, located at Xq28 with a complex genomic organisation. It contains 26 exons spanning 186kb of genomic DNA, and produces a 9kb transcript, resulting in a functional protein of 2332 amino acids. Over 900 mutations, which span a wide variety of categories, including rearrangements; complete or partial gene deletions; large insertions; duplications; frameshift mutations; splicing defects; nonsense and missense mutations, have been identified in the FVIII gene. Most mutations are rare or family specific, except for the intron 22 inversion mutation, which is reported to account for 45-50% of mutations in severe haemophilia A patients in most populations. A second inversion mutation, in intron 1, accounts for approximately 3.8% of haemophilia A patients in the UK. In South Africa, diagnostic mutation testing is currently only available for the intron 22 inversion mutation. Linked marker analysis is used to track high risk alleles in families where the disease-causing mutation is unknown. This study aims to evaluate an mRNA-based method to identify disease-causing mutations in South African haemophilia A patients and improve the diagnostic service. Blood samples from 120 patients were tested first for the intron 22 and then for intron 1 inversion mutations. Inversion negative patients were analysed further using mRNA. A mutation has been identified in 73.3% (88/120) of all patients. 30% (36/120) of patients had the intron 22 inversion, 2.5% (3/120) an intron 1 inversion and 40.8% (49/120) of patients had a mutation identified by mRNA analysis. A mutation was not identified in the remaining 26.7% (32/120) due to sample and technical difficulties. Of the 49 mutations identified through mRNA analysis, 28 patients (57.1%) have a point mutation (17 missense (34.7%), 9 nonsense (18.4%) and 2 splice-site mutations (4.1%)), 9 patients (18.4%) have a deletion and 7 patients (14.3%) have an insertion. Another 5 patients (10.2%) have a complex mutation (including patients where an exon deletion was detected on mRNA analysis, but no mutation was identified on DNA analysis). One mutation, c.3637insA, was found recurrently in 14% (6/43) of patients from the white population. This single base insertion results in a frameshift mutation with a premature stop codon at amino acid 1221 (only translating about half the normal FVIII protein). This common mutation, together with haplotype analysis, suggests a founder effect for this mutation. mRNA analysis of the FVIII gene is a novel technique in mutation detection for haemophilia A. It decreases the costs involved in sequencing the coding region and it offers improved mutation detection compared to DNA analysis. Diagnostic testing in South Africa should be extended from the current intron 22 inversion mutation to include DNA analysis for the intron 1 inversion and the founder mutation (c.3637insA) in white patients, followed by mRNA testing, starting with the analysis of the fragments spanning exon 14. mRNA analysis identifies an additional 55.7% of mutations compared to conventional diagnostic testing for the intron 22 inversion alone. en_US
dc.language.iso en en_US
dc.subject haemophilia A en_US
dc.subject mutation detection en_US
dc.subject South Africa en_US
dc.title Improved mutation detection for haemophilia A in South Africa en_US
dc.type Thesis en_US

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