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 language | English |
|---|---|
| Pages (from-to) | 55-64 |
| Number of pages | 10 |
| Journal | Indian Journal of Human Genetics |
| Volume | 14 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - Jul 1 2008 |
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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 journal › Article
}
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 -