Title

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

Document Type

Article

Publication Date

7-1-2008

Comments

This article has been peer reviewed. It was published in: Indian Journal of Human Genetics.

Volume 14, Issue 2, 1 July 2008, Pages 55-64.

The published version is available at http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2840789/. DOI: 10.4103/0971-6866.44106

Copyright © Indian Journal of Human Genetics

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.