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Factor IX mutations in hemophilia B patients from Liaoning Province, China

Dear Sir or Madam, We investigated the molecular basis of hemophilia B (HB) in 27 patients with HB from 23 families residing in Liaoning Province, China, and found a variety of mutations that cause mild-to-severe HB. The patients were recruited from No. 202 Hospital of PLA, Shenyang. The diagnosis was based on factor IX levels, as determined by a one-stage clotting assay performed on citrated, platelet-poor plasma. Eleven patients had mild HB (factor IX coagulam activity: 6~30%), eight had moderate HB (factor IX coagulam activity: 2~5%), and eight had severe HB (factor IX coagulam activity: <1%). Thirteen patients had a family history of HB. The research was approved by the institutional review board of our hospital (No. 202 Hospital of PLA, Shenyang), and informed consent was obtained from all participants. Genomic DNA was extracted from the peripheral blood leukocytes of the 27 index cases using a DNA extraction kit (Tiangen, China). F9 exons were amplified in a total of seven reactions using the polymerase chain reaction (PCR). Exons 1–8 of the F9 gene were sequenced in all patients. The F9 gene sequence was applied as a reference, and mutations were numbered accordingly the NCBI reference (NM_000133.3(cDNA)).We also utilized the Webbased program, SWISS-MODEL (http://swissmodel.expasy.org/ ), to predict the tertiary structure of the normal and mutated proteins. We found that each patient had only one-amino acid alteration that was considered a candidate disease-causing mutation. These putative mutations were not found as second-site changes in the other hemophiliac patients or as neutral polymorphisms in 100 healthy individuals of the same ethnicity. Screening for molecular changes in the F9 gene among members of the 23 families in this study revealed 19 point mutations, of which three were novel (c.485 G>T in family 23, c.1191 insT in family 8, and c.1331 A>G in family 19; Table 1). The basic predicted tertiary structures of sequence fragments of the normal and mutated proteins are shown in Figure 1. The change of structures may result in the change of protein function, and further cause HB. Some report showed that HB occurs in approximately 1 in every 30 000 male live births [1], and this rate varies little within races. HB is invariably caused by the mutations in the F9 gene, and these mutations show considerable heterogeneity among different cohorts of the patients with HB [2–4]. HB is an X-linked, inherited, blood clotting disorder. It is characterized by a factor IX deficiency caused by the mutations in the F9 gene located at Xq27.1 (GenBank Accession Number: K02402.1). Hepatocytes synthesize a factor IX precursor containing an amino-terminal signal sequence and a propeptide, both of which are cleaved off in separate reactions prior to protein secretion; defects in the removal of these sequences result in a nonfunctional protein [5]. The F9 gene and factor IX protein have shown considerable sequence homology and near-identical structural organization to those of the vitamin Kdependent serine proteases coagulation factor VII, factor X, and protein C. The F9 mutations detected in the patients with HB have been associated with varying disease severity [6]. The current factor IX mutation database includes 1094 unique F9 variants, with 798 point mutations [7]. We systematically analyzed the sequence of the entire F9 gene in 23 apparently unrelated families and identified causative mutations in each family. A total of 19 different mutations were detected, including one frameshift mutation, one nonsense point mutation, two splice-site point mutations, and fifteen missense mutations. Point mutations (single-nucleotide substitutions) were the most common gene defects. This result is consistent with another study, which reported that point mutations are present in approximately 73% of patients with HB [8]. CG transitions can cause missense or nonsense mutations, depending on the codon involved and the types of transition (C-to-T or G-to-A). C-to-T transitions are particularly damaging at arginine codons of the CGA type, because they involve the translation stop codon TGA (a nonsense mutation). In this study, eight point mutations involved CG sites, indicating that the CG dinucleotide is a mutational hot spot in the F9 gene, in agreement with another study [9].