Identification of FVIII gene mutations in patients with hemophilia A using new combinatorial sequencing by hybridization

M. Chetta, A. Drmanac, R. Santacroce, E. Grandone, S. Surrey, P. Fortina, M. Margaglione

Research output: Contribution to journalArticle

Abstract

Background: Standard methods of mutation detection are time consuming in Hemophilia A (HA) rendering their application unavailable in some analysis such as prenatal diagnosis. Objectives: To evaluate the feasibility of combinatorial sequencing-by-hybridization (cSBH) as an alternative and reliable tool for mutation detection in FVIII gene. Patients/Methods: 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 deficiency. Results: 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. Conclusions: cSBH is a reliable tool for mutation detection in FVIII gene and may represent a complementary method for the genetic screening of HA patients.

Original languageEnglish
Pages (from-to)55-64
Number of pages10
JournalIndian Journal of Human Genetics
Volume14
Issue number2
DOIs
Publication statusPublished - Jul 1 2008

Keywords

  • Combinatorial sequencing-by-hybridization
  • FVIII gene
  • Hemophilia A

ASJC Scopus subject areas

  • Genetics
  • Genetics(clinical)

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