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Identification Of Fviii Gene Mutations In Patients Withhemophilia A Using New Combinatorial Sequencing By Hybridization | 6082
ISSN: 2155-9872
Journal of Analytical & Bioanalytical Techniques
Open Access
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Standard methods of mutation detection are time consuming in Hemophilia A
(HA) rendering their application unavailable in some analysis such as prenatal
diagnosis. To evaluate the feasibility of combinatorial sequencing-by-hybridization
(cSBH) as an alternative and reliable tool for mutation detection in FVIII gene. We
have applied a new method of cSBH that uses two different colors for detection
of multiple point mutations in the FVIII gene. The 26 exons encompassing the HA
gene were analyzed in 7 newly diagnosed Italian patients and in 19 previously
characterized individuals with FVIII de� ciency. Data show that, when solution-
phase TAMRA and QUASAR labeled 5-mer oligonucleotide sets mixed with
unlabeled target PCR templates are co-hybridized in the presence of DNA ligase to
universal 6-mer oligonucleotide probe-based arrays, a number of mutations can be
successfully detected. The technique was reliable also in identifying a mutant FVIII
allele in an obligate heterozygote. A novel missense mutation (Leu1843Thr) in exon
16 and three novel neutral polymorphisms are presented with an updated protocol
for 2-color cSBH.cSBH is a reliable tool for mutation detection in FVIII gene and may
represent a complementary method for the genetic screening of HA patients.
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