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

2 Citations (Scopus)

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

Fingerprint

Hemophilia A
Mutation
Genes
Exons
Color
DNA Ligases
Oligonucleotide Probes
Genetic Testing
Missense Mutation
Heterozygote
Prenatal Diagnosis
Point Mutation
Oligonucleotides
Alleles
Polymerase Chain Reaction

Keywords

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

ASJC Scopus subject areas

  • Genetics
  • Genetics(clinical)

Cite this

Identification of FVIII gene mutations in patients with hemophilia A using new combinatorial sequencing by hybridization. / Chetta, M.; Drmanac, A.; Santacroce, R.; Grandone, E.; Surrey, S.; Fortina, P.; Margaglione, M.

In: Indian Journal of Human Genetics, Vol. 14, No. 2, 01.07.2008, p. 55-64.

Research output: Contribution to journalArticle

Chetta, M. ; Drmanac, A. ; Santacroce, R. ; Grandone, E. ; Surrey, S. ; Fortina, P. ; Margaglione, M. / Identification of FVIII gene mutations in patients with hemophilia A using new combinatorial sequencing by hybridization. In: Indian Journal of Human Genetics. 2008 ; Vol. 14, No. 2. pp. 55-64.
@article{495c87a30bd24bb0b0bd162220ed22d7,
title = "Identification of FVIII gene mutations in patients with hemophilia A using new combinatorial sequencing by hybridization",
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.",
keywords = "Combinatorial sequencing-by-hybridization, FVIII gene, Hemophilia A",
author = "M. Chetta and A. Drmanac and R. Santacroce and E. Grandone and S. Surrey and P. Fortina and M. Margaglione",
year = "2008",
month = "7",
day = "1",
doi = "10.4103/0971-6866.44106",
language = "English",
volume = "14",
pages = "55--64",
journal = "Indian Journal of Dermatology",
issn = "0019-5154",
publisher = "Medknow Publications and Media Pvt. Ltd",
number = "2",

}

TY - JOUR

T1 - Identification of FVIII gene mutations in patients with hemophilia A using new combinatorial sequencing by hybridization

AU - Chetta, M.

AU - Drmanac, A.

AU - Santacroce, R.

AU - Grandone, E.

AU - Surrey, S.

AU - Fortina, P.

AU - Margaglione, M.

PY - 2008/7/1

Y1 - 2008/7/1

N2 - 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.

AB - 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.

KW - Combinatorial sequencing-by-hybridization

KW - FVIII gene

KW - Hemophilia A

UR - http://www.scopus.com/inward/record.url?scp=56749181700&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=56749181700&partnerID=8YFLogxK

U2 - 10.4103/0971-6866.44106

DO - 10.4103/0971-6866.44106

M3 - Article

C2 - 20300295

AN - SCOPUS:56749181700

VL - 14

SP - 55

EP - 64

JO - Indian Journal of Dermatology

JF - Indian Journal of Dermatology

SN - 0019-5154

IS - 2

ER -