Efficient detection of factor IX mutations by denaturing high-performance liquid chromatography in Taiwanese hemophilia B patients, and the identification of two novel mutations

Abstract

Hemophilia B (HB) is an X-linked recessive disorder characterized by mutations in the clotting factor IX (FIX) gene that result in FIX deficiency. Previous studies have shown a wide variation of FIX gene mutations in HB. Although the quality of life in HB has greatly improved mainly because of prophylactic replacement therapy with FIX concentrates, there exists a significant burden on affected families and the medical care system. Accurate detection of FIX gene mutations is critical for genetic counseling and disease prevention in HB. In this study, we used denaturing high-performance liquid chromatography (DHPLC), which has proved to be a highly informative and practical means of detecting mutations, for the molecular diagnosis of our patients with HB. Ten Taiwanese families affected by HB were enrolled. We used the DHPLC technique followed by direct sequencing of suspected segments to detect FIX gene mutations. In all, 11 FIX gene mutations (8 point mutations, 2 small deletions/insertions, and 1 large deletion), including two novel mutations (exon6 c.687-695, del 9 mer and c.460-461, ins T) were found. According to the HB pedigrees, 25% and 75% of our patients were defined as familial and sporadic HB cases, respectively. We show that DHPLC is a highly sensitive and cost-effective method for FIX gene analysis and can be used as a convenient system for disease prevention.

Keywords: Denaturing high-performance liquid chromatography (DHPLC); Factor IX (FIX); Hemophilia B.

Figure 1

Algorithm of hemophilia B gene mutation analysis.

Figure 2

Sequences of two novel FIX gene mutations. (A) Segments containing putative mutations of Case No. 5 (female carrier) revealed to be heterozygous for exon6 c.687–695, del 9 mer and wild type. (B) Homozygous c.460–461, ins T in Case No. 11.

Figure 3

Nucleotides and amino acid sequences of the wild‐type and two novel mutant FIX genes. (A) Nucleotide positions 20518–20538 of the wild‐type FIX gene and 20529–20537 deletion (exon6 c.687–695, del 9 mer). The deleted nucleotides and the predicted mutated sequence are shown as red and green letters, respectively. (B) Nucleotide positions 17735–17758 of the wild‐type FIX gene and 17737–17738 insT (c.460–461, ins T). The insert T is indicated in red, and the predicted amino acids are shown as green letters.