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BACKGROUND Hemophilia B (H is a coagulation disorder with an X-linked recessive inheritance pattern, caused by plasma FIX deficiency. In Colombia, HB is considered a rare and high-cost disease, with 362 males reported in 2017. METHODS Here, we characterized 20 HB apparently unrelated families by PCR amplification and Sanger sequencing. RESULTS Fourteen unique variants were identified seven missense, three nonsense, one variant in the 3' UTR region, two large deletions 50 bp, and one intronic substitution that affects splicing c.52