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Clinical Chemistry 49: 815-818, 2003; 10.1373/49.5.815
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(Clinical Chemistry. 2003;49:815-818.)
© 2003 American Association for Clinical Chemistry, Inc.

Technical Briefs

Denaturing HPLC Procedure for Factor IX Gene Scanning

Giuseppe Castaldo1,2, Paola Nardiello1, Fabiana Bellitti1, Angiola Rocino3, Antonio Coppola4, Giovanni di Minno4 and Francesco Salvatore1,a

1 Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli "Federico II" and CEINGE-Biotecnologie Avanzate, I-80131 Napoli, Italy

2 Facoltà di Scienze, Università del Molise, 86100 Isernia, Italy

3 Centro Emofilia e Trombosi, Ospedale S.G. Bosco, 80141 Napoli, Italy

4 Centro di Coordinamento Regionale Emocoagulopatie, Dipartimento di Medicina Clinica e Sperimentale, Università di Napoli "Federico II", Napoli, Italy

aaddress correspondence to this author at: Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli "Federico II", via S. Pansini 5, I-80131 Naples, Italy; fax 39-081-746-3650, e-mail salvator@unina.it

The first 300 words of the full text of this article appear below.

Hemophilia B (HB) is an X-linked recessive bleeding disorder caused by mutations that produce factor IX (FIX) deficiency. The incidence of HB is ~1:30 000 live male births. The FIX gene spans ~34 kb and contains eight exons. The disease results from a myriad of mutations, and because of the rapid turnover of FIX mutations, there is no common mutation pattern in any ethnic group (1). Carrier and prenatal diagnosis can be made by linkage analysis (1)(2), which is rapid and inexpensive but limited by noninformative families, recombinant events, and the high incidence of germline mutations (1). Denaturing gradient gel electrophoresis (3)(4) and direct gene sequencing (5)(6)(7)(8) have been used for the direct identification of FIX mutations. Denaturing reversed-phase HPLC (D-HPLC), which has been used to scan several disease genes, is more sensitive than other scanning procedures and less expensive than direct sequencing (9)(10)(11)(12)(13). In addition, the post-PCR analysis can be automated.

We developed an original D-HPLC screening procedure for the whole FIX gene and analyzed a cohort of 18 unrelated HB patients from Southern Italy previously typed by direct sequencing. In all patients, diagnosis was confirmed by FIX assay (Table 1 ). The study was approved by the institutional ethics committee.


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  		Table 1. FIX mutations identified by direct sequencing in 18 unrelated patients with HB.

After obtaining informed consent, we collected blood samples by venipuncture at the time of sampling for routine molecular analyses and extracted the DNA with the "Nucleon" procedure (Amersham). Each DNA sample was amplified by PCR for all FIX exons and for the promoter region and then was analyzed by sequencing and by D-HPLC. FIX exons 2 . . . [Full Text of this Article]






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