Severe haemophilia B due to a 6 kb factor IX gene deletion including exon 4: Non-homologous recombination associated with a shortened transcript from whole blood

Summary

In genotyping a severe hemophilia B subject, exons 1–3 and 5–8 were normal. Exon 4 did not amplify, suggesting a partial gene deletion. Previously, a French family with an exon 4 deletion had severe haemophilia B with a circulating, dysfunctional factor IX protein missing its first growth factor-like domain; breakpoints were not analyzed. Using a 5’ primer for exon 3 and a 3’ primer for exon 5 fragments, the subject’s factor IX gene amplified a 5 kb fragment whereas 11 kb was predicted, indicating a 6 kb deletion. Restriction endonucleases localized the 3’ intron 4 deletion breakpoint to 1.2 kb 5’ to exon 5. Sequencing through the breakpoints revealed a 5,969 bp deletion that included exon 4 and was accompanied by a 13 bp duplication inserted near the 3’ breakpoint site. Haemophilia was familial; on testing, his mother was confirmed as a heterozygous carrier, whereas his sister was homozygous for the normal, larger fragments. As exons 4 and 5 of the factor IX gene are in frame, this deletion should produce a shortened transcript, missing 114 bp (38 codons from the first growth factor-like domain). Reverse transcription of mRNA prepared from whole blood and PCR identified the shorter cDNA fragment. Western blotting demonstrated a smaller factor IX protein.

Keywords

Deletion breakpoints - ectopic mRNA - exon 4 - factor IX gene - haemophilia B

• References

• 1 Green PM, Giannelli F, Sommer SS. et al. Haemophilia B Mutation Database Version 13: http://www.kcl.ac.uk/ip/petergreen/haemBdatabase.html.2004
• 2 Vidaud M, Chabret C, Gazengel C. et al. A de novo intragenic deletion of the potential EGF domain of the factor IX gene in a family with severe hemophilia B. Blood 1986; 68: 961-963.
• 3 Yoshitake S, Schach BG, Foster DC. et al. Nucleotide sequence of the gene for human factor IX (antihemophilic factor B). Biochemistry 1985; 24: 3736-3750.
• 4 Chen SH, Scott CR. Recombination between two 14-bp homologous sequences as the mechanism for gene deletion in factor IX Seattle 1. Am J Hum Genet 1990; 47: 1020-1022.
• 5 Bray GL, Thompson AR. Partial factor IX protein in a pedigree with hemophilia B due to a partial gene deletion. J Clin Invest 1986; 77: 1194-1200.
• 6 Ludwig M, Grimm T, Brackmann HH. et al. Parental origin of factor IX gene mutations, and their distribution in the gene. Am J Hum Genet 1992; 50: 164-173.
• 7 Ketterling RP, Vielhaber EL, Lind TJ. et al. The rates and patterns of deletions in the human factor IX gene. Am J Hum Genet 1994; 54: 201-213.
• 8 Li X, Drost JB, Roberts S. et al. Factor IX mutations in South Africans and African Americans are compatible with primarily endogenous influences upon recent germline mutations. Hum Mutat 2000; 16: 371.
• 9 Green PM, Bentley DR, Mibashan RS. et al. Partial deletion by illegitimate recombination of the factor IX gene in a haemophilia B family with two inhibitor patients. Mol Biol Med 1988; 5: 95-106.
• 10 Thompson AR. Molecular biology of the hemophilias. Prog Hemost Thromb 1991; 10: 175-214.
• 11 Green PM, Rowley G, Giannelli F. Unusual expression of the F9 gene in peripheral lymphocytes hinders investigation of F9 mRNA in hemophilia B patients. J Thromb Haemost 2003; 1: 2675-2676.
• 12 Cutler JA, Mitchell MJ, Savidge GF. More on: unusual expression of the F9 gene in peripheral lymphocytes hinders investigation of F9 mRNA in hemophilia B patients. J Thromb Haemost 2004; 2: 1021.
• 13 Van de Water NS, Tan T, May S. et al. Factor IX polypyrimidine tract mutation analysis using mRNA from peripheral blood leukocytes. J Thromb Haemost 2004; 2: 2073-2075.
• 14 Thompson AR, Chen SH. Characterization of factor IX defects in hemophilia B patients. Methods Enzymol 1993; 222: 143-169.
• 15 Bajaj SP, Thompson AR. Molecular and Structural biology of factor IX. In: Hemostasis and Thrombosis: Basic Principles and Clinical Practice. 5th ed. Lippincott Williams & Wilkins, Philadelphia; 2006; 131-150.
• 16 Zhong D, Smith KJ, Birktoft JJ. et al. First epidermal growth factor-like domain of human blood coagulation factor IX is required for its activation by factor VIIa/tissue factor but not by factor XIa. Proc Natl Acad Sci USA 1994; 91: 3574-3578.
• 17 Abeysinghe SS, Chuzhanova N, Krawczak M. et al. Translocation and gross deletion breakpoints in human inherited disease and cancer I: Nucleotide composition and recombination-associated motifs. Hum Mutat 2003; 22: 229-244.
• 18 Liu Q, Li X, Chen JS. et al. Robust dosage-PCR for detection of heterozygous chromosomal deletions. Biotechniques 2003; 34: 558-562 565–566, 568 passim.
• 19 Pavlova A, Schroder J, Delev D. et al. Rapid and sensitive detection of heterozygous deletions of one or more exons in hemophilia A females by multiplex PCR and DHPLC technique. Haemophilia 2006; 12 (Suppl. 02) 11.