Description of the first mutation in the human tissue factor gene associated with a bleeding tendency

Abstract

In a recent publication in the Journal of Clinical Investigation, Schulman and colleagues discovered that an individual with an unexplained bleeding tendency had a heterozygous mutation in the tissue factor (TF) gene.1.Schulman S. El‐Darzi E. Florido M.H. et al.A coagulation defect arising from heterozygous premature termination of tissue factor.J Clin Invest. 2020; 130: 5302-5312https://doi.org/10.1172/JCI133780Crossref PubMed Scopus (7) Google Scholar Understanding the molecular basis of unexplained bleeding in patients is required to improve therapy and to expand our knowledge of the pathways involved in hemostasis. Individuals with unexplained bleeding disorders in the National Institute for Health Research BioResource Rare Diseases Program of the United Kingdom 100 000 genome project were subjected to whole genome sequencing to identify potential causative genetic variants.2.Turro E. Astle W.J. Megy K. et al.Whole‐genome sequencing of patients with rare diseases in a national health system.Nature. 2020; 583: 96-102https://doi.org/10.1038/s41586‐020‐2434‐2Crossref PubMed Scopus (204) Google Scholar Of the 983 individuals with unexplained bleeding disorders, one individual was found to have a two‐nucleotide deletion in one allele of the TF gene, F3. The mutation introduced a premature stop codon that resulted in expression of a truncated form of TF (termed TFshort) missing the second fibronectin type III and transmembrane domains. Subsequent analysis revealed that the TFshort messenger RNA (mRNA) was subject to nonsense mediated decay, which is a translation‐dependent pathway that facilitates degradation of mRNAs containing premature stop codons.3.Kurosaki T. Popp M.W. Maquat L.E. Quality and quantity control of gene expression by nonsense‐mediated mRNA decay.Nat Rev Mol Cell Biol. 2019; 20: 406-420https://doi.org/10.1038/s41580‐019‐0126‐2Crossref PubMed Scopus (300) Google Scholar Furthermore, it was shown that TFshort protein had no significant procoagulant activity. This means that this individual was effectively heterozygous for functional TF having one allele generating wild‐type TF and one allele generating truncated nonfunctional TF. The individual had a history of menorrhagia, epistaxis, easy bruising, and an episode of bleeding following a dental extraction but was otherwise healthy. This work poses several interesting questions. Why has it taken so long to identify a mutation in the human TF gene leading to a loss of function? TF is unique in the coagulation cascade because it is the only coagulation factor that is not present in blood under normal conditions. Instead, TF is expressed by perivascular cells surrounding blood vessels and triggers coagulation after vascular injury.4.Grover S.P. Mackman N. Tissue factor: an essential mediator of hemostasis and trigger of thrombosis.Arterioscler Thromb Vasc Biol. 2018; 38: 709-725https://doi.org/10.1161/ATVBAHA.117.309846Crossref PubMed Scopus (328) Google Scholar Therefore, unlike circulating coagulation factors, a deficiency of TF will not be detected with commonly used functional coagulation assays, such as the prothrombin time (PT) and the activated partial thromboplastin time (aPTT). Indeed, the PT uses exogenous TF as the activator, whereas the aPTT uses a negatively charged agent, such as kaolin, as the activator. The PT and/or aPTT are significantly prolonged in hemophilia and other rare inherited bleeding disorders, and these abnormal test results aid in the diagnosis of these coagulation factor deficiencies (Table 1).5.Palla R. Peyvandi F. Shapiro A.D. Rare bleeding disorders: diagnosis and treatment.Blood. 2015; 125: 2052-2061https://doi.org/10.1182/blood‐2014‐08‐532820Crossref PubMed Scopus (191) Google Scholar, 6.Mannucci P.M. Tuddenham E.G. The hemophilias–from royal genes to gene therapy.N Engl J Med. 2001; 344: 1773-1779https://doi.org/10.1056/NEJM200106073442307Crossref PubMed Scopus (802) Google Scholar As expected, Schulman and colleagues found that the heterozygous TF‐deficient individual had a normal PT and aPTT.1.Schulman S. El‐Darzi E. Florido M.H. et al.A coagulation defect arising from heterozygous premature termination of tissue factor.J Clin Invest. 2020; 130: 5302-5312https://doi.org/10.1172/JCI133780Crossref PubMed Scopus (7) Google Scholar Therefore, a gene sequencing approach was required to identify the loss‐of‐function mutation in the TF gene.TABLE 1Rare inherited bleeding disorders in coagulation proteinsFactorGeneDiseaseEstimated FrequencyGnomad pLOFFrequencyLaboratory Coagulation AbnormalitiesTFF3~1:25 000aEstimate from Gnomad database.1:28 291NoneFVIIF7~1:500 0001:12 860Prolonged PTFXIIF12~1:1 000 0001:8321Prolonged aPTTFXIF11Hemophilia C~1:1 000 0001:3823Prolonged aPTTFIXF9Hemophilia B~1:30 000bLive male births.NDProlonged aPTTFVIIIF8Hemophilia A~1:5000bLive male births.1:70 728Prolonged aPTTFXF10~1:1 000 0001:11 788Prolonged aPPTProlonged PTFVF5~1:1 000 0001:3450Prolonged aPTTProlonged PTProthrombinF2~1:1‐2 000 0001:12 860Prolonged aPTTProlonged PTFibrinogenFGA, FGB, FGGAfibrinogenemia~1:1 000 0001:3823Prolonged aPTTProlonged PTAbbreviations: ND, none detected; pLOF, probable loss of function.a Estimate from Gnomad database.b Live male births. Open table in a new tab Abbreviations: ND, none detected; pLOF, probable loss of function. What is the closest comparator to a heterozygous TF deficiency? FVII deficiency represents the most comparable rare inherited bleeding disorder given that TF serves as an essential cofactor for FVIIa.7.Mariani G. Herrmann F.H. Dolce A. et al.Clinical phenotypes and factor VII genotype in congenital factor VII deficiency.Thromb Haemost. 2005; 93: 481-487https://doi.org/10.1160/TH04‐10‐0650Crossref PubMed Scopus (181) Google Scholar, 8.Herrmann F.H. Wulff K. Auerswald G. Factor VII deficiency: clinical manifestation of 717 subjects from Europe and Latin America with mutations in the factor 7 gene.Haemophilia. 2009; 15: 267-280https://doi.org/10.1111/j.1365‐2516.2008.01910.xCrossref PubMed Scopus (118) Google Scholar FVII deficiency is a clinically heterogenous disorder with a broad clinical presentation.7.Mariani G. Herrmann F.H. Dolce A. et al.Clinical phenotypes and factor VII genotype in congenital factor VII deficiency.Thromb Haemost. 2005; 93: 481-487https://doi.org/10.1160/TH04‐10‐0650Crossref PubMed Scopus (181) Google Scholar, 8.Herrmann F.H. Wulff K. Auerswald G. Factor VII deficiency: clinical manifestation of 717 subjects from Europe and Latin America with mutations in the factor 7 gene.Haemophilia. 2009; 15: 267-280https://doi.org/10.1111/j.1365‐2516.2008.01910.xCrossref PubMed Scopus (118) Google Scholar Extrapolating from the clinical presentation of individuals with FVII deficiency, one would not necessarily expect increased bleeding in heterozygous TF deficient individuals with 50% levels of TF. For instance, only a minority (19%, 93/499) of individuals with heterozygous mutations in the FVII gene that resulted in pronounced reductions in FVII activity were symptomatic in a large‐scale analysis.8.Herrmann F.H. Wulff K. Auerswald G. Factor VII deficiency: clinical manifestation of 717 subjects from Europe and Latin America with mutations in the factor 7 gene.Haemophilia. 2009; 15: 267-280https://doi.org/10.1111/j.1365‐2516.2008.01910.xCrossref PubMed Scopus (118) Google Scholar This suggests that other factors, including mutations in other genes, could affect the hemostatic competency of individuals with FVII deficiency. Symptomatic patients with heterozygous FVII deficiency typically present with menorrhagia, epistaxis and easy bruising.7.Mariani G. Herrmann F.H. Dolce A. et al.Clinical phenotypes and factor VII genotype in congenital factor VII deficiency.Thromb Haemost. 2005; 93: 481-487https://doi.org/10.1160/TH04‐10‐0650Crossref PubMed Scopus (181) Google Scholar, 8.Herrmann F.H. Wulff K. Auerswald G. Factor VII deficiency: clinical manifestation of 717 subjects from Europe and Latin America with mutations in the factor 7 gene.Haemophilia. 2009; 15: 267-280https://doi.org/10.1111/j.1365‐2516.2008.01910.xCrossref PubMed Scopus (118) Google Scholar This pattern of bleeding is similar to that described for the individual with heterozygous TF deficiency.1.Schulman S. El‐Darzi E. Florido M.H. et al.A coagulation defect arising from heterozygous premature termination of tissue factor.J Clin Invest. 2020; 130: 5302-5312https://doi.org/10.1172/JCI133780Crossref PubMed Scopus (7) Google Scholar As with other bleeding disorders, symptomatic FVII deficiency has been categorized into three categories with different median levels of FVII (mild [14%], moderate [3.3%], and severe [1.4%]). As expected, patients with compound heterozygous and homozygous FVII deficiency have the lowest levels of FVII activity (Table 2).7.Mariani G. Herrmann F.H. Dolce A. et al.Clinical phenotypes and factor VII genotype in congenital factor VII deficiency.Thromb Haemost. 2005; 93: 481-487https://doi.org/10.1160/TH04‐10‐0650Crossref PubMed Scopus (181) Google Scholar, 8.Herrmann F.H. Wulff K. Auerswald G. Factor VII deficiency: clinical manifestation of 717 subjects from Europe and Latin America with mutations in the factor 7 gene.Haemophilia. 2009; 15: 267-280https://doi.org/10.1111/j.1365‐2516.2008.01910.xCrossref PubMed Scopus (118) Google Scholar One could speculate that there are as yet unidentified individuals with compound heterozygous or homozygous TF deficiency.TABLE 2Bleeding phenotypes in humans with FVII deficiencySeverityPhenotypeFVII Activity (%, Median ± IQR)Mutation Distribution (%, Het; Comp Het; Hom)Mild<2 bleeding symptomsaEpistaxis, Easy bruising, Gum bleeding, Muscle hematoma, Hematuria, Post‐operative bleeding, Menorrhagia.14 (3‐31)43; 29; 28Moderate>3 bleeding symptomsaEpistaxis, Easy bruising, Gum bleeding, Muscle hematoma, Hematuria, Post‐operative bleeding, Menorrhagia.3.3 (1‐22)16; 39; 45SevereCNS bleeding and/or GI bleeding and/or hemarthrosis with/without other bleeding1.4 (1‐3.8)2; 47; 5Adapted from7.Mariani G. Herrmann F.H. Dolce A. et al.Clinical phenotypes and factor VII genotype in congenital factor VII deficiency.Thromb Haemost. 2005; 93: 481-487https://doi.org/10.1160/TH04‐10‐0650Crossref PubMed Scopus (181) Google ScholarAbbreviations: CNS, central nervous system; comp het, compound heterozygous; GI, gastrointestinal; Het, heterozygous; hom, homozygous.a Epistaxis, Easy bruising, Gum bleeding, Muscle hematoma, Hematuria, Post‐operative bleeding, Menorrhagia. Open table in a new tab Adapted from7.Mariani G. Herrmann F.H. Dolce A. et al.Clinical phenotypes and factor VII genotype in congenital factor VII deficiency.Thromb Haemost. 2005; 93: 481-487https://doi.org/10.1160/TH04‐10‐0650Crossref PubMed Scopus (181) Google Scholar Abbreviations: CNS, central nervous system; comp het, compound heterozygous; GI, gastrointestinal; Het, heterozygous; hom, homozygous. Are mutations in other genes required to reveal a bleeding phenotype associated with heterozygous TF deficiency? The relationship between the identified F3 mutation that results in expression of TFshort and bleeding in the individual remains unclear. Importantly, although the mutation was inherited paternally a mild bleeding phenotype was also noted on the maternal side with menorrhagia and postpartum hemorrhages in the mother and mild bleeding symptoms in the aunt.1.Schulman S. El‐Darzi E. Florido M.H. et al.A coagulation defect arising from heterozygous premature termination of tissue factor.J Clin Invest. 2020; 130: 5302-5312https://doi.org/10.1172/JCI133780Crossref PubMed Scopus (7) Google Scholar This raises the possibility that additional mutations in other genes increase the penetrance of the observed F3 mutation. This finding would be consistent with contributions of additional disease modifying mutations in other genes that modulate the penetrance of F7 mutants. Incomplete penetrance has been observed in patients with von Willebrand disease, another rare bleeding disorder, that is also associated with a variable clinical phenotype.9.Swystun L.L. James P. Using genetic diagnostics in hemophilia and von Willebrand disease.Hematology Am Soc Hematol Educ Program. 2015; 2015: 152-159https://doi.org/10.1182/asheducation‐2015.1.152Crossref PubMed Scopus (13) Google Scholar Identification of additional cohorts with heterozygous loss‐of‐function F3 mutations is required to further investigate the causative nature of partial TF deficiency in the context of an unexplained bleeding tendency. How common are heterozygous TF variants likely to be? Schulman and colleagues used data from the Gnomad browser to provide an estimated frequency of probable loss‐of‐function (pLOF) mutations in F3. The Gnomad database is a repository of whole exome sequencing data from more than 140,000 individuals that identifies pLOF mutations based on a computational prediction model.10.Karczewski K.J. Francioli L.C. Tiao G. The mutational constraint spectrum quantified from variation in 141,456 humans.Nature. 2020; 581: 434-443https://doi.org/10.1038/s41586‐020‐2308‐7Crossref PubMed Scopus (3343) Google Scholar The estimated frequency of pLOF mutations in F3 was 1 in 28 291 individuals (Table 1). However, there is a marked disconnect between predictions of pLOF mutations in coagulation factor genes made using the Gnomad database and the frequency of coagulation factor deficiencies reported in the literature (Table 1).5.Palla R. Peyvandi F. Shapiro A.D. Rare bleeding disorders: diagnosis and treatment.Blood. 2015; 125: 2052-2061https://doi.org/10.1182/blood‐2014‐08‐532820Crossref PubMed Scopus (191) Google Scholar The Gnomad database excludes individuals with severe pediatric diseases and first‐degree relatives, likely including hemophilia A and B, which may explain why the estimated frequencies of F8 and F9 mutations are considerably lower in the database than reported in the literature. It is surprising that the predicted frequency of pLOF mutations in F3 is greater than that reported in the literature for hemophilia B and greater than the sum of all other rare inherited bleeding disorders (Table 1).5.Palla R. Peyvandi F. Shapiro A.D. Rare bleeding disorders: diagnosis and treatment.Blood. 2015; 125: 2052-2061https://doi.org/10.1182/blood‐2014‐08‐532820Crossref PubMed Scopus (191) Google Scholar, 6.Mannucci P.M. Tuddenham E.G. The hemophilias–from royal genes to gene therapy.N Engl J Med. 2001; 344: 1773-1779https://doi.org/10.1056/NEJM200106073442307Crossref PubMed Scopus (802) Google Scholar Gene size is thought to be an important determinant of the expected number of loss‐of‐function mutations and potentially the corresponding disease frequency. The F8 gene encodes the 330kDa FVIII protein that is six times larger than the FIX protein encoded by the F9 gene. Correspondingly, the reported frequency of hemophilia A, caused by FVIII deficiency, is six times greater than that of hemophilia B, caused by FIX deficiency. It would, therefore, be expected that the frequency of TF deficiency would be lower than that of FVII deficiency based on the size of the respective proteins, 33 and 50 kDa. However, the expected frequency for TF deficiency, based on estimates from the Gnomad database, is markedly higher (20 times) than that reported in the literature for FVII deficiency. Further work is required to more accurately determine the frequency of disease‐causing mutations in the TF gene. How can patients with TF deficiency be identified in the future? A number of genetic bleeding disorder panels have been developed that use gene sequencing to identify variants in genes associated with rare bleeding disorders.11.Bastida J.M. Del Rey M. Lozano M.L. et al.Design and application of a 23‐gene panel by next‐generation sequencing for inherited coagulation bleeding disorders.Haemophilia. 2016; 22: 590-597https://doi.org/10.1111/hae.12908Crossref PubMed Scopus (45) Google Scholar, 12.Simeoni I. Stephens J.C. Hu F. et al.A high‐throughput sequencing test for diagnosing inherited bleeding, thrombotic, and platelet disorders.Blood. 2016; 127: 2791-2803https://doi.org/10.1182/blood‐2015‐12‐688267Crossref PubMed Scopus (138) Google Scholar Surprisingly, F3 is one of the only coagulation factor genes not routinely included in these panels. F3 is perhaps one of the best candidates for identification of disease‐causing variants by gene sequencing given that classical laboratory coagulation tests are not altered. The work of Schulman and colleagues supports the inclusion of F3 in genetic bleeding disorder panels. Importantly, inclusion of F3 in these panels may identify additional mutations in patients with unexplained bleeding tendencies and would improve our understanding of the relationship between F3 variants and bleeding. The TF‐FVIIa complex is responsible for idling of the coagulation cascade.13.Mackman N. The role of tissue factor and factor VIIa in hemostasis.Anesth Analg. 2009; 108: 1447-1452https://doi.org/10.1213/ane.0b013e31819bceb1Crossref PubMed Scopus (228) Google Scholar One study found that inhibition of TF in chimpanzees reduced basal levels of the FIX and FX activation peptides.14.ten Cate H. Bauer K.A. Levi M. et al.The activation of factor X and prothrombin by recombinant factor VIIa in vivo is mediated by tissue factor.J Clin Invest. 1993; 92: 1207-1212https://doi.org/10.1172/JCI116691Crossref PubMed Google Scholar In addition, basal levels of thrombin‐antithrombin complexes in plasma of mice are decreased in mice expressing low levels of TF.15.Pawlinski R. Pedersen B. Schabbauer G. et al.Role of tissue factor and protease‐activated receptors in a mouse model of endotoxemia.Blood. 2004; 103: 1342-1347https://doi.org/10.1182/blood‐2003‐09‐3051Crossref PubMed Scopus (245) Google Scholar These results suggest that, in theory, measurement of activation peptides and coagulation markers in plasma could be used to detect a functional deficient in TF in individuals with unexplained bleeding. What is the bleeding phenotype associated with TF and FVII‐deficient mice? Mice with a complete deficiency for TF are not viable and die during embryonic development or shortly after birth.16.Toomey J.R. Kratzer K.E. Lasky N.M. Stanton J.J. Broze G.J. Targeted disruption of the murine tissue factor gene results in embryonic lethality.Blood. 1996; 88: 1583-1587Crossref PubMed Google Scholar, 17.Carmeliet P. Mackman N. Moons L. et al.Role of tissue factor in embryonic blood vessel development.Nature. 1996; 383: 73-75https://doi.org/10.1038/383073a0Crossref PubMed Scopus (581) Google Scholar, 18.Bugge T.H. Xiao Q. Kombrinck K.W. et al.Fatal embryonic bleeding events in mice lacking tissue factor, the cell‐associated initiator of blood coagulation.Proc Natl Acad Sci U S A. 1996; 93: 6258-6263Crossref PubMed Scopus (288) Google Scholar Similarly, FVII‐null mice have a lethal hemostatic defect.19.Rosen E.D. Chan J.C. Idusogie E. et al.Mice lacking factor VII develop normally but suffer fatal perinatal bleeding.Nature. 1997; 390: 290-294https://doi.org/10.1038/36862Crossref PubMed Scopus (186) Google Scholar Importantly, TF‐null mice can be rescued by the expression of a low level (~1%) of human TF from a human TF minigene.20.Parry G.C. Erlich J.H. Carmeliet P. Luther T. Mackman N. Low levels of tissue factor are compatible with development and hemostasis in mice.J Clin Invest. 1998; 101: 560-569https://doi.org/10.1172/JCI814Crossref PubMed Scopus (181) Google Scholar These low TF mice survive to wean at close to the expected frequency but exhibit spontaneous fatal hemorrhages later in life.21.Pedersen B. Holscher T. Sato Y. Pawlinski R. Mackman N. A balance between tissue factor and tissue factor pathway inhibitor is required for embryonic development and hemostasis in adult mice.Blood. 2005; 105: 2777-2782https://doi.org/10.1182/blood‐2004‐09‐3724Crossref PubMed Scopus (91) Google Scholar, 22.Grover S.P. Schmedes C.M. Auriemma A.C. et al.Differential roles of factors IX and XI in murine placenta and hemostasis under conditions of low tissue factor.Blood Adv. 2020; 4: 207-216https://doi.org/10.1182/bloodadvances.2019000921Crossref PubMed Scopus (4) Google Scholar Low TF mice demonstrate a mildly prolonged bleeding in the tail transection and saphenous laser injury models.23.Pawlinski R. Pedersen B. Erlich J. Mackman N. Role of tissue factor in haemostasis, thrombosis, angiogenesis and inflammation: lessons from low tissue factor mice.Thromb Haemost. 2004; 92: 444-450https://doi.org/10.1160/TH04‐05‐0309Crossref PubMed Scopus (77) Google Scholar, 24.Getz T.M. Piatt R. Petrich B.G. Monroe D. Mackman N. Bergmeier W. Novel mouse hemostasis model for real‐time determination of bleeding time and hemostatic plug composition.J Thromb Haemost. 2015; 13: 417-425https://doi.org/10.1111/jth.12802Crossref PubMed Scopus (43) Google Scholar It is notable that mice expressing very low levels of FVII have a similar phenotype to low TF mice.25.Rosen E.D. Xu H. Liang Z. Martin J.A. Suckow M. Castellino F.J. Generation of genetically‐altered mice producing very low levels of coagulation factorVII.Thromb Haemost. 2005; 94: 493-497Crossref PubMed Scopus (38) Google Scholar, 26.Xu H. Noria F. Sandoval‐Cooper M.J. et al.Severe deficiency of coagulation factor VII results in spontaneous cardiac fibrosis in mice.J Pathol. 2009; 217: 362-371https://doi.org/10.1002/path.2454Crossref PubMed Scopus (11) Google Scholar Importantly, heterozygous TF mice and heterozygous FVII mice have been studied for more than 20 years and no spontaneous bleeding or hemostatic defects have been reported in either line of mice. For instance, no bleeding phenotype was apparent in mTF+/− mice subject to a tail vein transection model.16.Toomey J.R. Kratzer K.E. Lasky N.M. Stanton J.J. Broze G.J. Targeted disruption of the murine tissue factor gene results in embryonic lethality.Blood. 1996; 88: 1583-1587Crossref PubMed Google Scholar Surprisingly, Schulman and colleagues used the same line of mTF+/− mice and observed a bleeding phenotype in a tail amputation model. In addition, Schulman and colleagues observed impaired clot formation in mTF+/− mice subjected to severe, but not mild, injury in the cremaster arteriole laser injury model.1.Schulman S. El‐Darzi E. Florido M.H. et al.A coagulation defect arising from heterozygous premature termination of tissue factor.J Clin Invest. 2020; 130: 5302-5312https://doi.org/10.1172/JCI133780Crossref PubMed Scopus (7) Google Scholar Schulman and colleagues suggested that severe injuries are required to reveal a hemostatic defect associated with 50% levels of TF in mice. At present, it is unclear how these severe injuries in a mouse model relate to the hemostatic defects reported for the heterozygous TF individual. To conclude, this work provides the first evidence that a human F3 mutation is associated with a bleeding tendency. However, the results are based on one individual, and it is important to find additional heterozygous TF individuals with a bleeding tendency before we conclude that a 50% reduction in TF is associated with bleeding. The authors state that they have no relevant conflicts of interest. Steven P. Grover and Nigel Mackman jointly wrote the manuscript. Both authors have approved the final version. Dr. Grover is supported by a postdoctoral fellowship from the American Heart Association (19POST34370026). Dr. Mackman is supported by the John C. Parker Distinguished Professorship.American Heart Association19POST34370026