Denaturing HPLC Procedure for Factor IX Gene Scanning

[摘要] 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.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 to 7 were amplified using primers described elsewhere (5). Exon 1 and the upstream region of FIX (i.e., from the nucleotide −482), which …

[学科分类] 过敏症与临床免疫学