Factor IX Gene (F9) Genotyping Trends and Spectrum of Mutations Identified: A Reference Laboratory Experience

Abstract

In hemophilia B (HB), factor IX gene (F9) genotyping is used for molecular confirmation of affected individuals, for carrier testing, to facilitate the identification of those at risk for anaphylaxis/inhibitors (associated with large deletions), and to assist in assigning disease severity. Owing to test costs, optimal test utilization involves pre/post-test counseling and appropriate patient and test selection (e.g., mutation screening [F9MS] vs. known mutation [F9KM] testing). This article aims to review the trends and outcomes of F9-genotyping orders and describe the spectrum of variants identified in a sample of individuals in our reference laboratory. We performed a retrospective review of consecutive orders submitted to the Special Coagulation DNA Diagnostic Laboratory, Mayo Clinic, between 2012 and 2015. A total of 133 orders (38%) were identified for men: 118 (88%) were F9MS and 15 (12%) were F9KM. Thirteen orders (10%) were cancelled. A total of 209 orders were identified for women: 178 (85%) were F9MS and 31 (15%) were F9KM. Thirty-seven orders (18%) were cancelled and 30% of the tests performed yielded negative results. A total of 164 samples (47%) were received without clinical information. Seventeen previously unreported variants were identified. F9 genotyping provides useful information for HB management; however, 18% of our orders were cancelled and almost half were received without relevant clinical information, thus reaffirming the need for ongoing scrutiny of submitted orders. Optimal patient and test selection is important as is the accurate interpretation of variants identified. Most of the pathogenic variants identified were point mutations, with very few large deletions, consistent with the literature.

Keywords

hemophilia B - factor IX - genetic testing

• References

• 1 Mitchell M, Keeney S, Goodeve A. ; UK Haemophilia Centre Doctors' Organization Haemophilia Genetics Laboratory Network. The molecular analysis of haemophilia B: a guideline from the UK haemophilia centre doctors' organization haemophilia genetics laboratory network. Haemophilia 2005; 11 (04) 398-404
• 2 Sharathkumar A, Hardesty B, Greist A. , et al. Variability in bleeding phenotype in Amish carriers of haemophilia B with the 31008 C-->T mutation. Haemophilia 2009; 15 (01) 91-100
• 3 Toulon P, Berruyer M, Brionne-François M. , et al. Age dependency for coagulation parameters in paediatric populations. Results of a multicentre study aimed at defining the age-specific reference ranges. Thromb Haemost 2016; 116 (01) 9-16
• 4 Bowen DJ. Haemophilia A and haemophilia B: molecular insights. Mol Pathol 2002; 55 (02) 127-144
• 5 Simioni P, Tormene D, Tognin G. , et al. X-linked thrombophilia with a mutant factor IX (factor IX Padua). N Engl J Med 2009; 361 (17) 1671-1675
• 6 Chu K, Wu SM, Stanley T, Stafford DW, High KA. A mutation in the propeptide of Factor IX leads to warfarin sensitivity by a novel mechanism. J Clin Invest 1996; 98 (07) 1619-1625
• 7 Radic CP, Rossetti LC, Abelleyro MM. , et al. Assessment of the F9 genotype-specific FIX inhibitor risks and characterisation of 10 novel severe F9 defects in the first molecular series of Argentinian patients with haemophilia B. Thromb Haemost 2013; 109 (01) 24-33
• 8 Thorland EC, Drost JB, Lusher JM. , et al. Anaphylactic response to factor IX replacement therapy in haemophilia B patients: complete gene deletions confer the highest risk. Haemophilia 1999; 5 (02) 101-105
• 9 Briët E, Bertina RM, van Tilburg NH, Veltkamp JJ. Hemophilia B Leyden: a sex-linked hereditary disorder that improves after puberty. N Engl J Med 1982; 306 (13) 788-790
• 10 de Brasi C, El-Maarri O, Perry DJ, Oldenburg J, Pezeshkpoor B, Goodeve A. Genetic testing in bleeding disorders. Haemophilia 2014; 20 (04) (Suppl. 04) 54-58
• 11 Perry DJ, Cumming T, Goodeve A. , et al. The UK National External Quality Assessment Scheme for heritable bleeding disorders. Semin Thromb Hemost 2014; 40 (02) 261-268
• 12 Burke W, Pinsky LE, Press NA. Categorizing genetic tests to identify their ethical, legal, and social implications. Am J Med Genet 2001; 106 (03) 233-240
• 13 Richards S, Aziz N, Bale S. , et al; ACMG Laboratory Quality Assurance Committee. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 2015; 17 (05) 405-424
• 14 Costa JM, Ernault P, Vidaud D, Vidaud M, Meyer D, Lavergne JM. Fast and efficient mutation detection method using multiplex PCR and cycle sequencing--application to haemophilia B. Thromb Haemost 2000; 83 (02) 244-247
• 15 Kaiser RJ, MacKellar SL, Vinayak RS, Sanders JZ, Saavedra RA, Hood LE. Specific-primer-directed DNA sequencing using automated fluorescence detection. Nucleic Acids Res 1989; 17 (15) 6087-6102
• 16 Rydz N, Leggo J, Tinlin S, James P, Lillicrap D. The Canadian “National Program for hemophilia mutation testing” database: a ten-year review. Am J Hematol 2013; 88 (12) 1030-1034
• 17 Alabek M, Mohan R, Raia M. Genetic counselling for hemophilia. In: Treatment of Hemophilia. Montréal: World Federation of Hemophilia; 2015
• 18 Belvini D, Salviato R, Radossi P. , et al; AICE HB Study Group. Molecular genotyping of the Italian cohort of patients with hemophilia B. Haematologica 2005; 90 (05) 635-642