Medknow Publications on behalf of Indian Society 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