M.Sc. (Med.), Faculty of Health Sciences, University of the Witwatersrand, 2009. / 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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/7410 |
| Date | 03 November 2009 |
| Creators | Mitchell, Claire Lynne |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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