Complete F9 Gene Deletion, Duplication, and Triplication Rearrangements: Implications for Factor IX Expression and Clinical Phenotypes

Abstract

Background Factor IX (FIX) plays a critical role in blood coagulation. Complete deletion of F9 results in severe hemophilia B, whereas the clinical implications of complete F9 duplication and triplication remain understudied.

Objective To investigate the rearrangement mechanisms underlying complete F9 deletion (cases 1 and 2), duplication (cases 3 and 4), and triplication (case 5), and to explore their association with FIX expression levels and clinical impacts.

Methods Plasma FIX levels were detected using antigen and activity assays. CNVplex technology, optical genome mapping, and long-distance polymerase chain reaction were employed to characterize the breakpoints of the chromosomal rearrangements.

Results Cases 1 and 2 exhibited FIX activities below 1%. Case 3 displayed FIX activities within the reference range. However, cases 4 and 5 showed a significant increase in FIX activities. Alu-mediated nonallelic homologous recombination was identified as the cause of F9 deletion in case 1; FoSTeS/MMBIR (Fork Stalling and Template Switching/microhomology-mediated break-induced replication) contributed to both F9 deletion and tandem duplication observed in cases 2 and 3; BIR/MMBIR (break-induced replication/microhomology-mediated break-induced replication) mediated by the same pair of low-copy repeats results in similar duplication–triplication/inversion–duplication (DUP–TRP/INV–DUP) rearrangements in cases 4 and 5, leading to complete F9 duplication and triplication, respectively.

Conclusion Large deletions involving the F9 gene exhibit no apparent pattern, and the extra-hematologic clinical phenotypes require careful analysis of other genes within the deletion. The impact of complete F9 duplication and triplication on FIX expression might depend on the integrity of the F9 upstream sequence and the specific rearrangement mechanisms. Notably, DUP–TRP/INV–DUP rearrangements significantly elevate FIX activity and are closely associated with thrombotic phenotypes.

Keywords

factor IX - genomic structural variation - hemophilia B - low-copy repeats - thrombosis

• References

• 1 Smith SA, Travers RJ, Morrissey JH. How it all starts: initiation of the clotting cascade. Crit Rev Biochem Mol Biol 2015; 50 (04) 326-336
• 2 Berntorp E, Fischer K, Hart DP. et al. Haemophilia. Nat Rev Dis Primers 2021; 7 (01) 45
• 3 Khachidze M, Buil A, Viel KR. et al. Genetic determinants of normal variation in coagulation factor (F) IX levels: genome-wide scan and examination of the FIX structural gene. J Thromb Haemost 2006; 4 (07) 1537-1545
• 4 van Hylckama Vlieg A, van der Linden IK, Bertina RM, Rosendaal FR. High levels of factor IX increase the risk of venous thrombosis. Blood 2000; 95 (12) 3678-3682
• 5 Teixeira Mello TB, Machado TF, Montavão SA, Ozello MC, Annichino-Bizzacchi JM. Assessing the coagulation factor levels, inherited thrombophilia, and ABO blood group on the risk for venous thrombosis among Brazilians. Clin Appl Thromb Hemost 2009; 15 (04) 408-414
• 6 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
• 7 Wu W, Xiao L, Wu X. et al. Factor IX alteration p.Arg338Gln (FIX Shanghai) potentiates FIX clotting activity and causes thrombosis. Haematologica 2021; 106 (01) 264-268
• 8 Xu Z, Spencer HJ, Harris VA, Perkins SJ. An updated interactive database for 1692 genetic variants in coagulation factor IX provides detailed insights into hemophilia B. J Thromb Haemost 2023; 21 (05) 1164-1176
• 9 Chan V, Au P, Lau P, Chan TK. A novel haemophilia B defect due to partial duplication of the factor IX gene. Br J Haematol 1994; 86 (03) 601-609
• 10 Wheeler RB, Cutler JA, Alamelu J, Mitchell MJ. The first report of a multi-exon duplication in the F9 gene causative of severe haemophilia B. Haemophilia 2015; 21 (05) e433-e435
• 11 Xie X, Chen C, Liang Q. et al. Characterization of two large duplications of F9 associated with mild and severe haemophilia B, respectively. Haemophilia 2019; 25 (03) 475-483
• 12 Hol FA, Schepens MT, van Beersum SE. et al. Identification and characterization of an Xq26-q27 duplication in a family with spina bifida and panhypopituitarism suggests the involvement of two distinct genes. Genomics 2000; 69 (02) 174-181
• 13 Du C, Wang F, Li Z. et al. Xq26.3-q27.1 duplication including SOX3 gene in a Chinese boy with hypopituitarism: case report and two years treatment follow up. BMC Med Genomics 2022; 15 (01) 19
• 14 Kaminsky EB, Kaul V, Paschall J. et al. An evidence-based approach to establish the functional and clinical significance of copy number variants in intellectual and developmental disabilities. Genet Med 2011; 13 (09) 777-784
• 15 Radic CP, Abelleyro MM, Ziegler B. et al. Haemophilia B, severe childhood obesity and other extra-haematological features associated with similar 4Mb-deletions on Xq27: clinical findings, molecular insights and literature update. Haemophilia 2023; 29 (03) 844-854
• 16 Wu X, Lu Y, Ding Q. et al. Characterisation of large F9 deletions in seven unrelated patients with severe haemophilia B. Thromb Haemost 2014; 112 (03) 459-465
• 17 Nakamura Y, Ando Y, Takagi Y. et al. Distinct X chromosomal rearrangements in four haemophilia B patients with entire F9 deletion. Haemophilia 2016; 22 (03) 433-439
• 18 Onishi-Seebacher M, Korbel JO. Challenges in studying genomic structural variant formation mechanisms: the short-read dilemma and beyond. BioEssays 2011; 33 (11) 840-850
• 19 Mantere T, Kersten S, Hoischen A. Long-read sequencing emerging in medical genetics. Front Genet 2019; 10: 426
• 20 Yuan Y, Chung CY, Chan TF. Advances in optical mapping for genomic research. Comput Struct Biotechnol J 2020; 18: 2051-2062
• 21 Smith AC, Neveling K, Kanagal-Shamanna R. Optical genome mapping for structural variation analysis in hematologic malignancies. Am J Hematol 2022; 97 (07) 975-982
• 22 Fahiminiya S, Oikonomopoulos S, Rivard GE. et al. Deciphering a novel complex inversion affecting F8 in a family with severe haemophilia A by optical genome mapping. Haemophilia 2023; 29 (03) 921-924
• 23 Liu G, Sun J, Li Z, Chen Z, Wu W, Wu R. F9 mutations causing deletions beyond the serine protease domain confer higher risk for inhibitor development in hemophilia B. Blood 2023; 141 (06) 677-680
• 24 Li L, Wu X, Wu W, Ding Q, Cai X, Wang X. Clinical Manifestation and Mutation Spectrum of 53 Unrelated Pedigrees with Protein S Deficiency in China. Thromb Haemost 2019; 119 (03) 449-460
• 25 Cao P, Yang A, Wang R. et al. Germline duplication of SNORA18L5 increases risk for HBV-related hepatocellular carcinoma by altering localization of ribosomal proteins and decreasing levels of p53. Gastroenterology 2018; 155 (02) 542-556
• 26 Miranda-Furtado CL, Luchiari HR, Chielli Pedroso DC. et al. Skewed X-chromosome inactivation and shorter telomeres associate with idiopathic premature ovarian insufficiency. Fertil Steril 2018; 110 (03) 476-485.e1
• 27 Dardik R, Avishai E, Lalezari S. et al. Molecular mechanisms of skewed X-chromosome inactivation in female hemophilia patients-lessons from wide genome analyses. Int J Mol Sci 2021; 22 (16) 9074
• 28 Veyradier A, Boisseau P, Fressinaud E. et al; French Reference Center for von Willebrand disease. A laboratory phenotype/genotype correlation of 1167 French patients from 670 families with von Willebrand disease: a new epidemiologic picture. Medicine (Baltimore) 2016; 95 (11) e3038
• 29 Dittwald P, Gambin T, Gonzaga-Jauregui C. et al. Inverted low-copy repeats and genome instability–a genome-wide analysis. Hum Mutat 2013; 34 (01) 210-220
• 30 Zhang F, Khajavi M, Connolly AM, Towne CF, Batish SD, Lupski JR. The DNA replication FoSTeS/MMBIR mechanism can generate genomic, genic and exonic complex rearrangements in humans. Nat Genet 2009; 41 (07) 849-853
• 31 Burssed B, Zamariolli M, Bellucco FT, Melaragno MI. Mechanisms of structural chromosomal rearrangement formation. Mol Cytogenet 2022; 15 (01) 23
• 32 Lee JA, Carvalho CM, Lupski JR. A DNA replication mechanism for generating nonrecurrent rearrangements associated with genomic disorders. Cell 2007; 131 (07) 1235-1247
• 33 Carvalho CM, Ramocki MB, Pehlivan D. et al. Inverted genomic segments and complex triplication rearrangements are mediated by inverted repeats in the human genome. Nat Genet 2011; 43 (11) 1074-1081
• 34 Ishmukhametova A, Chen JM, Bernard R. et al. Dissecting the structure and mechanism of a complex duplication-triplication rearrangement in the DMD gene. Hum Mutat 2013; 34 (08) 1080-1084
• 35 Bahrambeigi V, Song X, Sperle K. et al. Distinct patterns of complex rearrangements and a mutational signature of microhomeology are frequently observed in PLP1 copy number gain structural variants. Genome Med 2019; 11 (01) 80
• 36 Li Y, Ding B, Mao Y, Zhang H, Wang X, Ding Q. Tandem and inverted duplications in haemophilia A: breakpoint characterisation provides insight into possible rearrangement mechanisms. Haemophilia 2023; 29 (04) 1121-1134
• 37 Anson DS, Blake DJ, Winship PR, Birnbaum D, Brownlee GG. Nullisomic deletion of the mcf.2 transforming gene in two haemophilia B patients. EMBO J 1988; 7 (09) 2795-2799
• 38 Jourdy Y, Chatron N, Carage ML. et al. Study of six patients with complete F9 deletion characterized by cytogenetic microarray: role of the SOX3 gene in intellectual disability. J Thromb Haemost 2016; 14 (10) 1988-1993
• 39 Stoof SCM, Kersseboom R, de Vries FAT, Kruip MJHA, Kievit AJA, Leebeek FWG. Hemophilia B in a female with intellectual disability caused by a deletion of Xq26.3q28 encompassing the F9. Mol Genet Genomic Med 2018; 6 (06) 1220-1224
• 40 Mason JA, Robertson JD. Extreme skewing of X-inactivation: rethinking severe haemophilia in women and girls. Haemophilia 2019; 25 (04) e286-e287
• 41 Sanyal A, Lajoie BR, Jain G, Dekker J. The long-range interaction landscape of gene promoters. Nature 2012; 489 (7414) 109-113
• 42 Yao L, Berman BP, Farnham PJ. Demystifying the secret mission of enhancers: linking distal regulatory elements to target genes. Crit Rev Biochem Mol Biol 2015; 50 (06) 550-573
• 43 Johnsen JM, Fletcher SN, Dove A. et al. Results of genetic analysis of 11 341 participants enrolled in the My Life, Our Future hemophilia genotyping initiative in the United States. J Thromb Haemost 2022; 20 (09) 2022-2034
• 44 Shen W, Gu Y, Zhu R, Zhang L, Zhang J, Ying C. Copy number variations of the F8 gene are associated with venous thromboembolism. Blood Cells Mol Dis 2013; 50 (04) 259-262
• 45 Van Laer C, Peerlinck K, Jacquemin M. et al. F11 gene duplication causes elevated FXI plasma levels and is a risk for venous thrombosis. Thromb Haemost 2022; 122 (06) 1058-1060
• 46 Megy K, Downes K, Morel-Kopp MC. et al. GoldVariants, a resource for sharing rare genetic variants detected in bleeding, thrombotic, and platelet disorders: communication from the ISTH SSC Subcommittee on Genomics in Thrombosis and Hemostasis. J Thromb Haemost 2021; 19 (10) 2612-2617
• 47 Turkut Tan T, Pariltay E, Avci Durmusaliogu E. et al. A unique case of thrombophilia: the role of F9 gene duplication and increased factor IX activity in cerebral venous thrombosis. J Thromb Haemost 2023; 21 (10) 2913-2916
• 48 Fang H, Deng X, Disteche CM. X-factors in human disease: impact of gene content and dosage regulation. Hum Mol Genet 2021; 30 (R2): R285-R295
• 49 Kujovich JL. Factor V Leiden thrombophilia. Genet Med 2011; 13 (01) 1-16
• 50 Rovida E, Merati G, D'Ursi P. et al. Identification and computationally-based structural interpretation of naturally occurring variants of human protein C. Hum Mutat 2007; 28 (04) 345-355