Haemophilia (2014), 20, e149–e156
DOI: 10.1111/hae.12346
ORIGINAL ARTICLE Genetics
Characterization of four novel molecular changes in the promoter region of the factor VIII gene C. NOUGIER,*† O. ROUALDES,*† M. FRETIGNY,* R. D’OIRON,‡ C. COSTA,§ C. NEGRIER*† and C . V I N C I G U E R R A * † *Service d’Hematologie Biologique, HCL, H^ opital Edouard Herriot; †EAM 4174 Hemostase, Inflammation et Sepsis, Universite Claude Bernard Lyon 1, Lyon; ‡Centre de Traitement pour Hemophiles, AP-HP H^ opital Bic^etre, Universite Paris XI, le Kremlin-Bic^etre; and §Departement de Genetique, CHU Henri Mondor-AP-HP, Creteil, France
Summary. Haemophilia A (HA) is an X-linked recessive bleeding disorder, caused by a wide variety of mutations in the factor VIII (F8) gene, leading to deficiency in the activity of coagulation FVIII. These mutations can affect all the F8 exons from the initiation codon to the termination codon, however, only few molecular changes in the promoter region of the F8 gene were reported so far. Here, we describe six nucleotide variations (c.-51G>A, c.-218T>C, c.-219C>T, c.219delC, c.-221T>A and c.-664G>A) detected in the F8 promoter and their correlation with clinical phenotype of the patients. Potential role of these mutations in HA was also assessed. Causality was demonstrated with transient transfection experiments using luciferase reporter gene plasmids and computational analysis. Two molecular changes (c.-51G>A and c.-664G>A) did not
seem to affect the promoter function of the F8 gene whereas c.-218T>C, c.-219C>T, c.-219delC, c.-221T>A mutations had an impact on the F8 promoter function and were responsible for HA. Furthermore, these mutations were associated with resistance to 1-deamino8-D-argininevasopressin (desmopressin) therapy when they were causative. When molecular variation was detected in F8 promoter, we propose to use prediction software and to verify predictions by reporter gene analysis. If the mutation is causative, it will be probably associated with a lack of therapeutic response to desmopressin and this clinical implication should be considered by clinicians.
Introduction
Since the F8 gene was cloned in 1984 [3], a wide diversity of genetic changes have been identified and more than 2000 causative mutations have been recorded in disease-specific databases (HADB database, http://hadb.org.uk/ June 2012; CHAMP Mutation List Database, www.cdc.gov/ncbddd/hemophilia/ champs.htlm). Genetic defects include the intron 22 and 1 inversions that account for nearly 45% and 5% of severe HA patients, respectively [4–7], as well as point mutations, insertions, duplications and deletions within the F8 gene coding and untranslated regions. Point mutations [missense, nonsense and messenger RNA (mRNA) splice-site mutations] represent approximately 70% of reported molecular defects in HA [8– 10] and are distributed all over the F8 exons from the initiation (codon +1) to the termination codon (+2352). The F8 is regulated by a 1175-bp promoter cloned in 1984 [3] and regulation of F8 expression involves a variety of transcription factors that bind to several partially identified regions within the promoter [10,11]. To date, a few single base substitutions have
Haemophilia A (HA, OMIM 306700) is an X-linked bleeding disorder caused by heterogeneous mutations in the factor VIII (FVIII) gene (F8) leading to partial or total deficiency of FVIII procoagulant activity (FVIII:C). The disease affects approximately 1 in 5000 men, and is classified as severe when residual plasma FVIII activity is less than 0.01 IU mL 1, moderate (0.01–0.05 IU mL 1) or mild (0.05–0.4 IU mL 1) [1]. The human F8 gene is located on the most distal band (Xq28) of the long arm of the X-chromosome and spans 186 kb of genomic DNA, with 26 exons of various sizes and 25 introns [2]. Correspondence: Christophe Nougier, Service d’Hematologie Biologique, Pav E, H^ opital Edouard Herriot, 5 Place d’Arsonval, 69437 Lyon Cedex 03, France. Tel.: +33 4 72 11 73 78; fax: +33 4 72 11 73 12; e-mail: [email protected] Accepted after revision 17 November 2013 © 2013 John Wiley & Sons Ltd
Keywords: factor VIII, haemophilia, mutation, promoter, transcription factors
e149
e150
C. NOUGIER et al.
been described in the 5′ flanking region of the F8 [12– 14] resulting in a binding site loss for transcription factor and a decreased F8 promoter activity responsible for mild HA with ineffective response to 1-deamino-8-D-arginine-vasopressin (desmopressin or DDAVP) treatment [15]. In this study, we describe four novel and two previously described nucleotide variations in the F8 gene promoter of 10 patients. To assess potential impact of these nucleotide variations on transcription factor binding sites, molecular changes were studied using two strategies including reporter gene system and bioinformatics approaches. Patients’ phenotype and response to DDAVP therapy were also examined.
by direct sequencing (ABI Prism 3130XL; Applied Biosystems, Saint Aubin, France) for exons showing abnormal patterns on dHPLC. When this procedure did not reveal any molecular change, coding regions, splices sites, the promoter and 3′ Untranslated region (UTR) regions of F8 were directly sequenced and large rearrangements were screened with multiplex ligationdependent probe amplification (MLPA) analysis using a kit for F8 (P178) (MRC-Holland, Amsterdam, the Netherlands). DNA of patient P1 was not checked for MLPA as the technique was not available 15 years ago when the genotype was performed; no further test was possible due to the loss of follow-up of the patient.
Construction of luciferase reporter gene constructs
Patients, materials and methods Patients We investigated nine unrelated patients with HA and one putative carrier female with nucleotide variation in the F8 promoter. Eight patients were suffering from mild HA (0.06–0.31 IU mL 1), one patient presented a severe HA (T substitution was found [13]. The genetic variations detected in patients P1 (c.51G>A), P2 (c.-218T>C) and P7 (c.-219delC), who presented a mild-HA phenotype, have never been described before. The c.-221T>A substitution was detected in a patient (P8) with a severe form of haemophilia. FVIII deficiency was also confirmed in this patient using the FVIII antigen assay (FVIII:Ag < 0.01 IU mL 1). The c.664G>A substitution, which has not been described in the literature, was not only detected in a patient (P10) with mild haemophilia (FVIII:C = 0.06 IU mL 1) but also in a pregnant woman (P9, 29 weeks of gestation), a putative carrier with a FVIII:C/VWF:Ag ratio of 0.54. No familial relationship between these two individuals was found. In addition, all these patients presented normal other phenotypic assays (VWF:Ag, VWF:RCo, VWF:FVIIIB, FV levels), which enabled us to rule out all other aetiologies that might have explained the reduced FVIII levels. In seven of the eight patients with phenotypes consistent with mild HA and tested for DDAVP response no increase in FVIII:C levels was found, although increased VWF levels were observed (Table 1). DDAVP testing was not indicated for patient P8 and P9 and not available for patient P5. Except for patient P8 with severe HA, the majority of the patients with mild HA experienced only very few bleeding
Table 1. F8 promoter gene variations and phenotypic results. Patient
Nucleotide variation
FVIII:C (IU mL 1)
FVIII:Ag (IU mL 1)
Reported clinical phenotype
Age at diagnosis (years)
Resistance to DDAVP therapy
Novel mutation (yes/no)
P1 P2 P3 P4 P5 P6 P7 P8 P9 P10
c.-51G>A c.-218T>C c.-219C>T
0.10 0.31 0.27 0.17 0.30 0.16 0.15 A
Yes Yes Yes
na, not available; DDAVP, desmopressin. DNA mutation numbering was based on cDNA sequence (RefSeq.M14113.1) according to international recommendations for the description of sequence changes given by the Human Genome Variation Society, HGVS (http://www.HGVS.org/mutnomen/).
© 2013 John Wiley & Sons Ltd
Haemophilia (2014), 20, e149--e156
e152
C. NOUGIER et al.
episodes as expected in relation to their specific FVIII:C levels (much less than one bleeding episode per year). Some patients were treated with recombinant or plasma-derived FVIII products for treatment or prevention of haemorrhages.
Analysis of sequence conservation Sequence alignment in the promoter region of the F8 gene between different species was performed using the ElDorado/Gene2Promoter programme. This comparison showed a degree of homology of 95% (Rhesus monkey), 99% (chimpanzee), 67% (rabbit), 69% (horse), 58% (cow), 54% (pig) and 57% (dog), respectively, with the analogous human sequence (GXP_107069). At position c.-51, an adenosine nucleotide was observed in Rhesus monkey, horse and cow indicating that the nucleotide variation c.-51G>A is probably not deleterious to Human (Fig. 1a). No nucleotide variation was observed among the seven studied species in terms of c.-218 and c.-219. How-
(a)
ever, one variation was identified at the c.-221 position, where thymine was substituted by guanine in monkeys (Fig. 1b). The c.-214 to c.-225 region showed relatively high conservation of the residues between species. At the c.-664 position, an adenosine nucleotide was also identified in rabbits, suggesting that the c.-664G>A substitution may be considered as probably benign (Fig. 1c).
Luciferase assays We used the reporter gene technique to establish whether these nucleotide variations were responsible for the observed phenotypes. Luminescence measurements obtained 48 h after transfection revealed a significantly greater signal for the WT promoter compared to the ‘negative control’ signal obtained for the plasmid without pGL4 promoter (P = 0.0043), thus proving that observed signal was specifically induced by the promoter inserted in the pGL4 vector (Fig. 2). Moreover, value of the relative luciferase activity for the non-transfected cells (‘background noise’ control) was below 4%. No statistically significant difference was noted between the c.-51G>A substitution (104%) and WT (P = 0.2403). As expected, the previously described c.219C>T substitution (P3, P4, P5, P6) caused a 65% decrease in luciferase expression compared to WT (P = 0.0022). The c.-218 T>C (P2), c.-219delC (P7) and c.-221T>A (P8) mutations led to 34%, 63% and 67% decreases in promoter activity respectively (Fig. 2). Although patient P8 had severe HA, the
(b)
(c)
Fig. 1. Alignment of F8 promoter sequences of different species. Alignment of the human, monkey, chimpanzee, rabbit, horse, cow, pig and dog fragment of the F8 promoter. (a) fragment c.-40 to c.-60 including the nucleotide c.-51; (b) fragment c.-208 to c.-227 including the nucleotides c.218, c.-219 and c.-221. (c) fragment c.-661 to c.-674 including the nucleotide c.-664.
Haemophilia (2014), 20, e149--e156
Fig. 2. Relative luciferase activity values for the F8 promoter variants. Results are expressed as a percentage of the activity observed for the wildtype sequence. NT, non-transfected cells; pGL4 = promoterless reporter plasmid. *P < 0.05.
© 2013 John Wiley & Sons Ltd
NUCLEOTIDE VARIATIONS IN F8 PROMOTER REGION
e153
Fig. 3. Diagram of the F8 gene promoter and the probable transcription factor binding sites. The region (-208 to -227) containing the studied mutations was analysed in P-Match. WT, wild-type sequence. The nucleotide variations c.-219C>T, c.-219delC and c.-221T>A led to a binding site loss for transcription factor c/EBPb. The nucleotide variations c.-218T>C and c.-221T>A led to create a binding site for transcription factor YY1.
c.-221T>A mutation did not totally abolish the promoter activity (70% of decrease). Signal obtained with the c.-664G>A substitution (105%) was not significantly different from WT signal (P = 1.000) (Fig. 2).
Study of transcription factor binding site Comparative analysis of the WT sequence and the mutated sequences using P-Match also allowed identification of transcription factor binding sites on the promoter. Comparison of the native sequence with the mutated sequences showed that the nucleotide variations c.-219C>T, c.-219delC and c.-221T>A led to a binding site loss for transcription factor c/enhancerbinding proteins (EBP)b (Fig. 3). In addition, c.218T>C and c.-221T>A substitutions led to the appearance of a binding site for YY1 (Yin Yang 1) transcription factor (Fig. 3). However, analysis of the mutated sequences in P-Match showed that c.-51G>A and c.-664G>A substitutions did not affect any transcription factor binding sites.
Discussion Since description of the F8 promoter region in 1984 by Gitschier et al., 10 nucleotide variations have been reported (Table 2). Responsibility of these mutations in the cause of HA was demonstrated for c.-219C>T, c-255A>G and c.-257T>G mutations, however, some of these substitutions are unlikely to affect the expression of F8 and may represent polymorphisms. In this study, we describe five new nucleotide variations (c.-51G>A, c.-218T>C, c.-219delC, c.-221T>A, c.-664G>A) in the promoter region of the F8 gene. © 2013 John Wiley & Sons Ltd
We also studied the c.-219C>T mutation already studied by several teams (Table 2). Zimmerman et al. showed this mutation to be responsible for a 94% reduction in promoter activity in a cellular model using the HepG2 line [13,14]. In this study, PLC/PRF/ 5 cells were used to obtain a higher expression of F8 promoter activity and, therefore, of the reporter gene. This has been confirmed by the fact that FVIII mRNA is present in PLC cells but not in HepG2 cells [11,18]. In PLC/PRF/5 cells, the c.-219C>T mutation indeed had a lower impact on promoter activity (70% decrease) compared to the WT. These results are consistent with patients FVIII:C levels (0.17– 0.31 IU mL 1) as well as data by Dai et al., indicating this mutation accounts for a mild form of HA [13]. In the same region, the new mutations c.-218T>C, c.-219delC and c.-221T>A led to a significant decrease in promoter activity, thus demonstrating the role of this region in F8 gene transcription. Any substitution or deletion at these positions may compromise the binding of one or several transcription factors, leading to a decreased FVIII synthesis. Since description of the F8 gene promoter region, binding sites for different transcription factors such as hepatocyte nuclear factors 1 and 4 (NF1 and NF4), nuclear factor jB (NF-jB), CCAAT/EBP (C/EBP) and NFY transcription factors [19] have been identified. A particular region that spans from c.-279 to c.-64 has been considered as essential for an effective promoter activity and would be able to interact with c/EBPa and c/EBPb transcription factors ( 275 to 249) [11]. McGlynn et al. described a binding site within this region ( 233 to 216) for an unidentified transcription factor [10]. Further investigations on c.-219C>T mutation using Haemophilia (2014), 20, e149--e156
e154
C. NOUGIER et al.
Table 2. Summary of reported F8 promoter missense mutations. Nucleotide change
FVIII:C (IU mL 1)
HADB
c.-25A>G c.-42C>T c.-112G>A
na Mild 0.40
No Yes No
c.-126C>T c.-219C>T
c.-255A>G c.-257T>G c.-257T>A c.-257T>C c.-860A>G
0.50 0.31 0.30 na 0.22 0.29 0.28 0.21 na
No No
No No No
No
Studied by cells expression
Reference
No No No No Yes (HepG2 cells, HEK293 cells) No Yes (HepG2 cells, SK-HEP-1 cells) No Yes (HepG2 cells, HEK293 cells) In PLC/PRF/5 cells Yes (HepG2 cells, HEK293 cells) No Yes (HepG2 cells, HEK293 cells)
[33] HADB [12] [34] [14] [35] [12] [34] [14] [13] [14] [14,15,28] [14]
No
[36]
na, not available. All identified genomic variations were screened against HADB (http://hadb.org.uk/; Accessed June 2012).
the electrophoretic mobility shift assay provided evidence for causality of this mutation and its role in disruption of a transcription factor binding site. This site is contained in PLC/PRF/5 nuclear extracts and is located from 208 to 228 [14]. So, it is quite likely that c.-218T>C, c.-219delC, c.-221T>A mutations described in this study affect this region and may cause a transcription defect responsible for mild HA forms. Interspecies comparison of the c-215 to c-225 region also showed a strong homology, indicating the essential role of this region in the expression of promoter activity (Fig. 1b). Comparative analysis of the WT sequence and the mutated sequences using P-Match software also enabled to identify some transcription factor binding sites on the promoter. Comparison of the native sequence with the mutated sequences indeed showed that c.-219C>T, c.-219delC and c.-221T>A mutations led to a binding site loss for c/EBPb transcription factor (Fig. 3). This is consistent with the previous description of a region containing binding sites for transcription factors in the c/EBP class between 267 and 148 (regions D and C) [11]. In addition, c.218T>C and c.-221T>A substitutions led to the appearance of a binding site for YY1 transcription factor (Fig. 3). YY1 (also known as NF-E1 or UCRBP) is an ubiquitous transcription factor of the zinc finger proteins family. It regulates genes involved in the development and differentiation of cells and cell cycle. This transcription factor plays as a transcriptional repressor in a number of cellular promoters [20,21]. The appearance of a binding site for this transcription factor has been previously described as possibly causing disease [22], and might be partly responsible for the mild HA of the patient with the c.218T>C mutation. The c.-221T>A mutation has been shown to cause the disappearance of an activator site (C/EBPb) and the appearance of a repressor site (YY1) resulting in a reduced F8 expression. Although our Haemophilia (2014), 20, e149--e156
results obtained with the P-Match software are consistent with current data for c/EBPb transcription factor, potential impact of YY1 transcription factor on the F8 promoter activity needs to be confirmed. The c.-221T>A mutation cannot per se explain the severity of the patient P8. To our knowledge, no mutation in the F8 promoter region has been yet described as causing a severe HA. However, no other genetic abnormalities were detected in the F8 coding regions, at F8 splicing sites or in the 3′ UTR for this patient. Therefore, another genetic event is likely to be responsible for the severity of the HA diagnosed in this patient. In addition, no decrease in luciferase expression and therefore in promoter activity was detected with c.-51G>A or c.664G>A substitutions. The c.-664G>A substitution may not be responsible for the mild HA found in patient P10 or for the discrepant FVIII/VWF ratio found in patient P9 (Table 1). This is consistent with previous studies, demonstrating that any deletion in the region from c.-776 to c.-386 does not lead to a decreased F8 promoter activity [11]. This result was confirmed for substitution c.-664G>A by the analysis in Alamutâ (Interactive Biosoftware, Rouen, France) indicating that this substitution should be considered as an single nucleotide polymorphisms (SNP) (SNP rs4898404). Analysis of the mutated sequences in P-Match showed that substitutions c.-51G>A and c.-664G>A do not affect any transcription factor binding sites. Moreover, the interspecies comparison also showed that there are comparable nucleotide variations in c.-51 and c.-664, confirming that c.-51G>A and c.-664G>A substitutions may be considered as SNP. Still we were unsuccessful in determining the genetic defects in P1, P9 and P10 patients. These mutations we report in the F8 promoter region also seem to be associated with absence of response to DDAVP. Administration of DDAVP that has been the preferred treatment for mild haemophilia and type 1 VWD since the beginning of the 1980s, © 2013 John Wiley & Sons Ltd
NUCLEOTIDE VARIATIONS IN F8 PROMOTER REGION
usually increases FVIII:C levels twofold to sixfold [23,24]. Recently, a number of parameters including the molecular abnormality causing HA have been shown to influence DDAVP response [25,26]. In some cases, this treatment can be inadequate (partial response) or ineffective (no response) [28]. In this study, seven patients with a promoter mutation did not respond effectively to this treatment. There are no data for the remaining three patients. This is consistent with the study by Riccardi et al., demonstrating that the c.-257T>G mutation was associated with ineffective DDAVP therapy [15,27]. However, this resistance was also observed in 8 of 10 patients without F8 mutations [28]. DDAVP releases the VWF and FVIII contained in endothelial cells [29] but it is difficult to explain this lack of response, as the mechanisms causing DDAVP to mobilize FVIII are currently poorly understood. Our knowledge of promoter regions has greatly improved over the last 20 years. Numerous mutations identified in the promoters of other genes have been previously studied (factor IX and haemophilia B Leyden [30], protein C [31], haptoglobin [32] and explain the patients’ clinical phenotypes. However, molecular variations in the F8 promoter are rare and represent less than 0.1% of cases of HA [13,14]. These variations are not necessarily all causative and it is therefore necessary to prove their role in the onset of the disease. In this
References 1 White GC II, Rosendaal F, Aledort LM, Lusher JM, Rothschild C, Ingerslev J. Definitions in hemophilia. Recommendation of the scientific subcommittee on factor VIII and factor IX of the scientific and standardization committee of the International Society on Thrombosis and Haemostasis. Thromb Haemost 2001; 85: 560. 2 Vehar GA, Keyt B, Eaton D et al. Structure of human factor VIII. Nature 1984; 312: 337–42. 3 Gitschier J, Wood WI, Goralka TM et al. Characterization of the human factor VIII gene. Nature 1984; 312: 326–30. 4 Lakich D, Kazazian HH Jr, Antonarakis SE, Gitschier J. Inversions disrupting the factor VIII gene are a common cause of severe haemophilia A. Nat Genet 1993; 5: 236–41. 5 Antonarakis SE, Rossiter JP, Young M et al. Factor VIII gene inversions in severe hemophilia A: results of an international consortium study. Blood 1995; 86: 2206–12. 6 Bagnall RD, Waseem N, Green PM, Giannelli F. Recurrent inversion breaking intron 1 of the factor VIII gene is a frequent cause of severe hemophilia A. Blood 2002; 99: 168–74. 7 Riccardi F, Tagliaferri A, Manotti C, Pattacini C, Neri TM. Intron factor VIII gene inversion in a population of Italian hemophilia A patients. Blood 2002; 100: 3432.
© 2013 John Wiley & Sons Ltd
e155
study, only four of six nucleotide variations detected were considered as causative for HA. When molecular variation was detected in F8 promoter, we propose to use prediction software and to verify predictions by reporter gene analysis. If the mutation is causative, it will be probably associated with a lack of therapeutic response to DDAVP and this clinical implication should be considered by clinicians.
Acknowledgements Many thanks to Dr. Caron de Fromentel for the gift of the PLC/PRF/5 cell lines. This study would not have been possible without the help of Dorothee Pellechia, Dr Bertrand M, Dr. Monpoux F, Dr Roussel Robert, Dr Pierre Louis, Dr Lienhart A and Dr Meunier S, Dr Peynet and Dr Durand B. The authors thank Dr Enjolras N and Dr Plantier JL for their excellent scientific assistance.
Author contributions C. Nougier, O. Roualdes, M. Fretigny and C. Vinciguerra performed the research, C. Nougier and O. Roualdes, C. Costa, R. d’Oiron analysed the data and wrote the paper. C. Negrier, C. Vinciguerra, C. Nougier designed the research study.
Disclosures The authors stated that they have no interests that might be perceived as posing a conflict or bias.
8 Goodeve AC, Peake IR. The molecular basis of hemophilia A: genotype–phenotype relationships and inhibitor development. Semin Thromb Hemost 2003; 29: 23–30. 9 Ogata K, Selvaraj SR, Miao HZ, Pipe SW. Most factor VIII B domain missense mutations are unlikely to be causative mutations for severe hemophilia A: implications for genotyping. J Thromb Haemost 2011; 9: 1183–90. 10 McGlynn LK, Mueller CR, Begbie M, Notley CR, Lillicrap D. Role of the liverenriched transcription factor hepatocyte nuclear factor 1 in transcriptional regulation of the factor VIII gene. Mol Cell Biol 1996; 16: 1936–45. 11 Figueiredo MS, Brownlee GG. cis-acting elements and transcription factors involved in the promoter activity of the human factor VIII gene. J Biol Chem 1995; 270: 11828–38. 12 Bogdanova N, Markoff A, Eisert R et al. Spectrum of molecular defects and mutation detection rate in patients with mild and moderate hemophilia A. Hum Mutat 2007; 28: 54–60. 13 Dai L, Cutler JA, Savidge GF, Mitchell MJ. Characterization of a causative mutation of hemophilia A identified in the promoter region of the factor VIII gene (F8). J Thromb Haemost 2008; 6: 193–5. 14 Zimmermann MA, Meier D, Oldenburg J, Muller CR, Rost S. Identification and characterization of mutations in the promoter
15
16
17
18
19
20
21
region of the factor VIII gene. J Thromb Haemost 2012; 10: 314–7. Riccardi F, Rivolta GF, Franchini M, Pattacini C, Neri TM, Tagliaferri A. Characterization of a novel mutation in the F8 promoter region associated with mild hemophilia A and resistance to DDAVP therapy. J Thromb Haemost 2009; 7: 1234–5. Bagnall RD, Giannelli F, Green PM. Int22 h-related inversions causing hemophilia A: a novel insight into their origin and a new more discriminant PCR test for their detection. J Thromb Haemost 2006; 4: 591–8. Chekmenev DS, Haid C, Kel AE. P-Match: transcription factor binding site search by combining patterns and weight matrices. Nucleic Acids Res 2005; 33: W432–7. Wion KL, Kelly D, Summerfield JA, Tuddenham EG, Lawn RM. Distribution of factor VIII mRNA and antigen in human liver and other tissues. Nature 1985; 317: 726–9. Graw J, Brackmann HH, Oldenburg J, Schneppenheim R, Spannagl M, Schwaab R. Haemophilia A: from mutation analysis to new therapies. Nat Rev Genet 2005; 6: 488–501. Shi Y, Lee JS, Galvin KM. Everything you have ever wanted to know about Yin Yang 1. Biochim Biophys Acta 1997; 1332: F49– 66. Yokoyama NN, Pate KT, Sprowl S, Waterman ML. A role for YY1 in repression of
Haemophilia (2014), 20, e149--e156
e156
22
23
24
25
26
27
C. NOUGIER et al.
dominant negative LEF-1 expression in colon cancer. Nucleic Acids Res 2010; 38: 6375–88. Li JR, Li JG, Deng GH et al. A common promoter variant of TBX21 is associated with allele specific binding to Ying-Yang 1 and reduced gene expression. Scand J Immunol 2011; 73: 449–58. Mannucci PM. Desmopressin (DDAVP) for treatment of disorders of hemostasis. Prog Hemost Thromb 1986; 8: 19–45. Castaman G. Desmopressin for the treatment of haemophilia. Haemophilia 2008; 14(Suppl. 1): 15–20. Mannucci PM. Desmopressin (DDAVP) in the treatment of bleeding disorders: the first twenty years. Haemophilia 2000; 6(Suppl. 1): 60–7. Seary ME, Feldman D, Carcao MD. DDAVP responsiveness in children with mild or moderate haemophilia A correlates with age, endogenous FVIII:C level and with haemophilic genotype. Haemophilia 2012; 18: 50–5. Castaman G, Mancuso ME, Giacomelli SH et al. Molecular and phenotypic determi-
Haemophilia (2014), 20, e149--e156
28
29
30
31
nants of the response to desmopressin in adult patients with mild hemophilia A. J Thromb Haemost 2009; 7: 1824–31. Tagliaferri A, Di Perna C, Riccardi F, Pattacini C, Rivolta GF, Franchini M. The natural history of mild haemophilia: a 30year single centre experience. Haemophilia 2012; 18: 166–74. Xu L, Nichols TC, Sarkar R, McCorquodale S, Bellinger DA, Ponder KP. Absence of a desmopressin response after therapeutic expression of factor VIII in hemophilia A dogs with liver-directed neonatal gene therapy. Proc Natl Acad Sci U S A 2005; 102: 6080–5. Hirosawa S, Fahner JB, Salier JP, Wu CT, Lovrien EW, Kurachi K. Structural and functional basis of the developmental regulation of human coagulation factor IX gene: factor IX Leyden. Proc Natl Acad Sci U S A 1990; 87: 4421–5. Berg LP, Scopes DA, Alhaq A, Kakkar VV, Cooper DN. Disruption of a binding site for hepatocyte nuclear factor 1 in the protein C gene promoter is associated with hereditary thrombophilia. Hum Mol Genet 1994; 3: 2147–52.
32 Grant DJ, Maeda N. A base substitution in the promoter associated with the human haptoglobin 2-1 modified phenotype decreases transcriptional activity and responsiveness to interleukin-6 in human hepatoma cells. Am J Hum Genet 1993; 52: 974–80. 33 Margaglione M, Castaman G, Morfini M et al. The Italian AICE-Genetics hemophilia A database: results and correlation with clinical phenotype. Haematologica 2008; 93: 722–8. 34 Green P, Bagnall R, Waseem N, Giannelli F. Haemophilia A mutations in the UK: results of screening one-third of the population. Br J Haematol 2008; 143: 115–28. 35 Lombardi AM, Zanon E, Sartori MT, Cabrio L, Vettore S, Girolami A. Molecular genetics of bleeding disorders. Haemophilia 2004; 10(Suppl. 3): 73–78. 36 Sanna V, Zarrilli F, Nardiello P et al. Mutational spectrum of F8 gene and prothrombotic gene variants in haemophilia A patients from Southern Italy. Haemophilia 2008; 14: 796–803.
© 2013 John Wiley & Sons Ltd
DOI: 10.1111/hae.12346
ORIGINAL ARTICLE Genetics
Characterization of four novel molecular changes in the promoter region of the factor VIII gene C. NOUGIER,*† O. ROUALDES,*† M. FRETIGNY,* R. D’OIRON,‡ C. COSTA,§ C. NEGRIER*† and C . V I N C I G U E R R A * † *Service d’Hematologie Biologique, HCL, H^ opital Edouard Herriot; †EAM 4174 Hemostase, Inflammation et Sepsis, Universite Claude Bernard Lyon 1, Lyon; ‡Centre de Traitement pour Hemophiles, AP-HP H^ opital Bic^etre, Universite Paris XI, le Kremlin-Bic^etre; and §Departement de Genetique, CHU Henri Mondor-AP-HP, Creteil, France
Summary. Haemophilia A (HA) is an X-linked recessive bleeding disorder, caused by a wide variety of mutations in the factor VIII (F8) gene, leading to deficiency in the activity of coagulation FVIII. These mutations can affect all the F8 exons from the initiation codon to the termination codon, however, only few molecular changes in the promoter region of the F8 gene were reported so far. Here, we describe six nucleotide variations (c.-51G>A, c.-218T>C, c.-219C>T, c.219delC, c.-221T>A and c.-664G>A) detected in the F8 promoter and their correlation with clinical phenotype of the patients. Potential role of these mutations in HA was also assessed. Causality was demonstrated with transient transfection experiments using luciferase reporter gene plasmids and computational analysis. Two molecular changes (c.-51G>A and c.-664G>A) did not
seem to affect the promoter function of the F8 gene whereas c.-218T>C, c.-219C>T, c.-219delC, c.-221T>A mutations had an impact on the F8 promoter function and were responsible for HA. Furthermore, these mutations were associated with resistance to 1-deamino8-D-argininevasopressin (desmopressin) therapy when they were causative. When molecular variation was detected in F8 promoter, we propose to use prediction software and to verify predictions by reporter gene analysis. If the mutation is causative, it will be probably associated with a lack of therapeutic response to desmopressin and this clinical implication should be considered by clinicians.
Introduction
Since the F8 gene was cloned in 1984 [3], a wide diversity of genetic changes have been identified and more than 2000 causative mutations have been recorded in disease-specific databases (HADB database, http://hadb.org.uk/ June 2012; CHAMP Mutation List Database, www.cdc.gov/ncbddd/hemophilia/ champs.htlm). Genetic defects include the intron 22 and 1 inversions that account for nearly 45% and 5% of severe HA patients, respectively [4–7], as well as point mutations, insertions, duplications and deletions within the F8 gene coding and untranslated regions. Point mutations [missense, nonsense and messenger RNA (mRNA) splice-site mutations] represent approximately 70% of reported molecular defects in HA [8– 10] and are distributed all over the F8 exons from the initiation (codon +1) to the termination codon (+2352). The F8 is regulated by a 1175-bp promoter cloned in 1984 [3] and regulation of F8 expression involves a variety of transcription factors that bind to several partially identified regions within the promoter [10,11]. To date, a few single base substitutions have
Haemophilia A (HA, OMIM 306700) is an X-linked bleeding disorder caused by heterogeneous mutations in the factor VIII (FVIII) gene (F8) leading to partial or total deficiency of FVIII procoagulant activity (FVIII:C). The disease affects approximately 1 in 5000 men, and is classified as severe when residual plasma FVIII activity is less than 0.01 IU mL 1, moderate (0.01–0.05 IU mL 1) or mild (0.05–0.4 IU mL 1) [1]. The human F8 gene is located on the most distal band (Xq28) of the long arm of the X-chromosome and spans 186 kb of genomic DNA, with 26 exons of various sizes and 25 introns [2]. Correspondence: Christophe Nougier, Service d’Hematologie Biologique, Pav E, H^ opital Edouard Herriot, 5 Place d’Arsonval, 69437 Lyon Cedex 03, France. Tel.: +33 4 72 11 73 78; fax: +33 4 72 11 73 12; e-mail: [email protected] Accepted after revision 17 November 2013 © 2013 John Wiley & Sons Ltd
Keywords: factor VIII, haemophilia, mutation, promoter, transcription factors
e149
e150
C. NOUGIER et al.
been described in the 5′ flanking region of the F8 [12– 14] resulting in a binding site loss for transcription factor and a decreased F8 promoter activity responsible for mild HA with ineffective response to 1-deamino-8-D-arginine-vasopressin (desmopressin or DDAVP) treatment [15]. In this study, we describe four novel and two previously described nucleotide variations in the F8 gene promoter of 10 patients. To assess potential impact of these nucleotide variations on transcription factor binding sites, molecular changes were studied using two strategies including reporter gene system and bioinformatics approaches. Patients’ phenotype and response to DDAVP therapy were also examined.
by direct sequencing (ABI Prism 3130XL; Applied Biosystems, Saint Aubin, France) for exons showing abnormal patterns on dHPLC. When this procedure did not reveal any molecular change, coding regions, splices sites, the promoter and 3′ Untranslated region (UTR) regions of F8 were directly sequenced and large rearrangements were screened with multiplex ligationdependent probe amplification (MLPA) analysis using a kit for F8 (P178) (MRC-Holland, Amsterdam, the Netherlands). DNA of patient P1 was not checked for MLPA as the technique was not available 15 years ago when the genotype was performed; no further test was possible due to the loss of follow-up of the patient.
Construction of luciferase reporter gene constructs
Patients, materials and methods Patients We investigated nine unrelated patients with HA and one putative carrier female with nucleotide variation in the F8 promoter. Eight patients were suffering from mild HA (0.06–0.31 IU mL 1), one patient presented a severe HA (T substitution was found [13]. The genetic variations detected in patients P1 (c.51G>A), P2 (c.-218T>C) and P7 (c.-219delC), who presented a mild-HA phenotype, have never been described before. The c.-221T>A substitution was detected in a patient (P8) with a severe form of haemophilia. FVIII deficiency was also confirmed in this patient using the FVIII antigen assay (FVIII:Ag < 0.01 IU mL 1). The c.664G>A substitution, which has not been described in the literature, was not only detected in a patient (P10) with mild haemophilia (FVIII:C = 0.06 IU mL 1) but also in a pregnant woman (P9, 29 weeks of gestation), a putative carrier with a FVIII:C/VWF:Ag ratio of 0.54. No familial relationship between these two individuals was found. In addition, all these patients presented normal other phenotypic assays (VWF:Ag, VWF:RCo, VWF:FVIIIB, FV levels), which enabled us to rule out all other aetiologies that might have explained the reduced FVIII levels. In seven of the eight patients with phenotypes consistent with mild HA and tested for DDAVP response no increase in FVIII:C levels was found, although increased VWF levels were observed (Table 1). DDAVP testing was not indicated for patient P8 and P9 and not available for patient P5. Except for patient P8 with severe HA, the majority of the patients with mild HA experienced only very few bleeding
Table 1. F8 promoter gene variations and phenotypic results. Patient
Nucleotide variation
FVIII:C (IU mL 1)
FVIII:Ag (IU mL 1)
Reported clinical phenotype
Age at diagnosis (years)
Resistance to DDAVP therapy
Novel mutation (yes/no)
P1 P2 P3 P4 P5 P6 P7 P8 P9 P10
c.-51G>A c.-218T>C c.-219C>T
0.10 0.31 0.27 0.17 0.30 0.16 0.15 A
Yes Yes Yes
na, not available; DDAVP, desmopressin. DNA mutation numbering was based on cDNA sequence (RefSeq.M14113.1) according to international recommendations for the description of sequence changes given by the Human Genome Variation Society, HGVS (http://www.HGVS.org/mutnomen/).
© 2013 John Wiley & Sons Ltd
Haemophilia (2014), 20, e149--e156
e152
C. NOUGIER et al.
episodes as expected in relation to their specific FVIII:C levels (much less than one bleeding episode per year). Some patients were treated with recombinant or plasma-derived FVIII products for treatment or prevention of haemorrhages.
Analysis of sequence conservation Sequence alignment in the promoter region of the F8 gene between different species was performed using the ElDorado/Gene2Promoter programme. This comparison showed a degree of homology of 95% (Rhesus monkey), 99% (chimpanzee), 67% (rabbit), 69% (horse), 58% (cow), 54% (pig) and 57% (dog), respectively, with the analogous human sequence (GXP_107069). At position c.-51, an adenosine nucleotide was observed in Rhesus monkey, horse and cow indicating that the nucleotide variation c.-51G>A is probably not deleterious to Human (Fig. 1a). No nucleotide variation was observed among the seven studied species in terms of c.-218 and c.-219. How-
(a)
ever, one variation was identified at the c.-221 position, where thymine was substituted by guanine in monkeys (Fig. 1b). The c.-214 to c.-225 region showed relatively high conservation of the residues between species. At the c.-664 position, an adenosine nucleotide was also identified in rabbits, suggesting that the c.-664G>A substitution may be considered as probably benign (Fig. 1c).
Luciferase assays We used the reporter gene technique to establish whether these nucleotide variations were responsible for the observed phenotypes. Luminescence measurements obtained 48 h after transfection revealed a significantly greater signal for the WT promoter compared to the ‘negative control’ signal obtained for the plasmid without pGL4 promoter (P = 0.0043), thus proving that observed signal was specifically induced by the promoter inserted in the pGL4 vector (Fig. 2). Moreover, value of the relative luciferase activity for the non-transfected cells (‘background noise’ control) was below 4%. No statistically significant difference was noted between the c.-51G>A substitution (104%) and WT (P = 0.2403). As expected, the previously described c.219C>T substitution (P3, P4, P5, P6) caused a 65% decrease in luciferase expression compared to WT (P = 0.0022). The c.-218 T>C (P2), c.-219delC (P7) and c.-221T>A (P8) mutations led to 34%, 63% and 67% decreases in promoter activity respectively (Fig. 2). Although patient P8 had severe HA, the
(b)
(c)
Fig. 1. Alignment of F8 promoter sequences of different species. Alignment of the human, monkey, chimpanzee, rabbit, horse, cow, pig and dog fragment of the F8 promoter. (a) fragment c.-40 to c.-60 including the nucleotide c.-51; (b) fragment c.-208 to c.-227 including the nucleotides c.218, c.-219 and c.-221. (c) fragment c.-661 to c.-674 including the nucleotide c.-664.
Haemophilia (2014), 20, e149--e156
Fig. 2. Relative luciferase activity values for the F8 promoter variants. Results are expressed as a percentage of the activity observed for the wildtype sequence. NT, non-transfected cells; pGL4 = promoterless reporter plasmid. *P < 0.05.
© 2013 John Wiley & Sons Ltd
NUCLEOTIDE VARIATIONS IN F8 PROMOTER REGION
e153
Fig. 3. Diagram of the F8 gene promoter and the probable transcription factor binding sites. The region (-208 to -227) containing the studied mutations was analysed in P-Match. WT, wild-type sequence. The nucleotide variations c.-219C>T, c.-219delC and c.-221T>A led to a binding site loss for transcription factor c/EBPb. The nucleotide variations c.-218T>C and c.-221T>A led to create a binding site for transcription factor YY1.
c.-221T>A mutation did not totally abolish the promoter activity (70% of decrease). Signal obtained with the c.-664G>A substitution (105%) was not significantly different from WT signal (P = 1.000) (Fig. 2).
Study of transcription factor binding site Comparative analysis of the WT sequence and the mutated sequences using P-Match also allowed identification of transcription factor binding sites on the promoter. Comparison of the native sequence with the mutated sequences showed that the nucleotide variations c.-219C>T, c.-219delC and c.-221T>A led to a binding site loss for transcription factor c/enhancerbinding proteins (EBP)b (Fig. 3). In addition, c.218T>C and c.-221T>A substitutions led to the appearance of a binding site for YY1 (Yin Yang 1) transcription factor (Fig. 3). However, analysis of the mutated sequences in P-Match showed that c.-51G>A and c.-664G>A substitutions did not affect any transcription factor binding sites.
Discussion Since description of the F8 promoter region in 1984 by Gitschier et al., 10 nucleotide variations have been reported (Table 2). Responsibility of these mutations in the cause of HA was demonstrated for c.-219C>T, c-255A>G and c.-257T>G mutations, however, some of these substitutions are unlikely to affect the expression of F8 and may represent polymorphisms. In this study, we describe five new nucleotide variations (c.-51G>A, c.-218T>C, c.-219delC, c.-221T>A, c.-664G>A) in the promoter region of the F8 gene. © 2013 John Wiley & Sons Ltd
We also studied the c.-219C>T mutation already studied by several teams (Table 2). Zimmerman et al. showed this mutation to be responsible for a 94% reduction in promoter activity in a cellular model using the HepG2 line [13,14]. In this study, PLC/PRF/ 5 cells were used to obtain a higher expression of F8 promoter activity and, therefore, of the reporter gene. This has been confirmed by the fact that FVIII mRNA is present in PLC cells but not in HepG2 cells [11,18]. In PLC/PRF/5 cells, the c.-219C>T mutation indeed had a lower impact on promoter activity (70% decrease) compared to the WT. These results are consistent with patients FVIII:C levels (0.17– 0.31 IU mL 1) as well as data by Dai et al., indicating this mutation accounts for a mild form of HA [13]. In the same region, the new mutations c.-218T>C, c.-219delC and c.-221T>A led to a significant decrease in promoter activity, thus demonstrating the role of this region in F8 gene transcription. Any substitution or deletion at these positions may compromise the binding of one or several transcription factors, leading to a decreased FVIII synthesis. Since description of the F8 gene promoter region, binding sites for different transcription factors such as hepatocyte nuclear factors 1 and 4 (NF1 and NF4), nuclear factor jB (NF-jB), CCAAT/EBP (C/EBP) and NFY transcription factors [19] have been identified. A particular region that spans from c.-279 to c.-64 has been considered as essential for an effective promoter activity and would be able to interact with c/EBPa and c/EBPb transcription factors ( 275 to 249) [11]. McGlynn et al. described a binding site within this region ( 233 to 216) for an unidentified transcription factor [10]. Further investigations on c.-219C>T mutation using Haemophilia (2014), 20, e149--e156
e154
C. NOUGIER et al.
Table 2. Summary of reported F8 promoter missense mutations. Nucleotide change
FVIII:C (IU mL 1)
HADB
c.-25A>G c.-42C>T c.-112G>A
na Mild 0.40
No Yes No
c.-126C>T c.-219C>T
c.-255A>G c.-257T>G c.-257T>A c.-257T>C c.-860A>G
0.50 0.31 0.30 na 0.22 0.29 0.28 0.21 na
No No
No No No
No
Studied by cells expression
Reference
No No No No Yes (HepG2 cells, HEK293 cells) No Yes (HepG2 cells, SK-HEP-1 cells) No Yes (HepG2 cells, HEK293 cells) In PLC/PRF/5 cells Yes (HepG2 cells, HEK293 cells) No Yes (HepG2 cells, HEK293 cells)
[33] HADB [12] [34] [14] [35] [12] [34] [14] [13] [14] [14,15,28] [14]
No
[36]
na, not available. All identified genomic variations were screened against HADB (http://hadb.org.uk/; Accessed June 2012).
the electrophoretic mobility shift assay provided evidence for causality of this mutation and its role in disruption of a transcription factor binding site. This site is contained in PLC/PRF/5 nuclear extracts and is located from 208 to 228 [14]. So, it is quite likely that c.-218T>C, c.-219delC, c.-221T>A mutations described in this study affect this region and may cause a transcription defect responsible for mild HA forms. Interspecies comparison of the c-215 to c-225 region also showed a strong homology, indicating the essential role of this region in the expression of promoter activity (Fig. 1b). Comparative analysis of the WT sequence and the mutated sequences using P-Match software also enabled to identify some transcription factor binding sites on the promoter. Comparison of the native sequence with the mutated sequences indeed showed that c.-219C>T, c.-219delC and c.-221T>A mutations led to a binding site loss for c/EBPb transcription factor (Fig. 3). This is consistent with the previous description of a region containing binding sites for transcription factors in the c/EBP class between 267 and 148 (regions D and C) [11]. In addition, c.218T>C and c.-221T>A substitutions led to the appearance of a binding site for YY1 transcription factor (Fig. 3). YY1 (also known as NF-E1 or UCRBP) is an ubiquitous transcription factor of the zinc finger proteins family. It regulates genes involved in the development and differentiation of cells and cell cycle. This transcription factor plays as a transcriptional repressor in a number of cellular promoters [20,21]. The appearance of a binding site for this transcription factor has been previously described as possibly causing disease [22], and might be partly responsible for the mild HA of the patient with the c.218T>C mutation. The c.-221T>A mutation has been shown to cause the disappearance of an activator site (C/EBPb) and the appearance of a repressor site (YY1) resulting in a reduced F8 expression. Although our Haemophilia (2014), 20, e149--e156
results obtained with the P-Match software are consistent with current data for c/EBPb transcription factor, potential impact of YY1 transcription factor on the F8 promoter activity needs to be confirmed. The c.-221T>A mutation cannot per se explain the severity of the patient P8. To our knowledge, no mutation in the F8 promoter region has been yet described as causing a severe HA. However, no other genetic abnormalities were detected in the F8 coding regions, at F8 splicing sites or in the 3′ UTR for this patient. Therefore, another genetic event is likely to be responsible for the severity of the HA diagnosed in this patient. In addition, no decrease in luciferase expression and therefore in promoter activity was detected with c.-51G>A or c.664G>A substitutions. The c.-664G>A substitution may not be responsible for the mild HA found in patient P10 or for the discrepant FVIII/VWF ratio found in patient P9 (Table 1). This is consistent with previous studies, demonstrating that any deletion in the region from c.-776 to c.-386 does not lead to a decreased F8 promoter activity [11]. This result was confirmed for substitution c.-664G>A by the analysis in Alamutâ (Interactive Biosoftware, Rouen, France) indicating that this substitution should be considered as an single nucleotide polymorphisms (SNP) (SNP rs4898404). Analysis of the mutated sequences in P-Match showed that substitutions c.-51G>A and c.-664G>A do not affect any transcription factor binding sites. Moreover, the interspecies comparison also showed that there are comparable nucleotide variations in c.-51 and c.-664, confirming that c.-51G>A and c.-664G>A substitutions may be considered as SNP. Still we were unsuccessful in determining the genetic defects in P1, P9 and P10 patients. These mutations we report in the F8 promoter region also seem to be associated with absence of response to DDAVP. Administration of DDAVP that has been the preferred treatment for mild haemophilia and type 1 VWD since the beginning of the 1980s, © 2013 John Wiley & Sons Ltd
NUCLEOTIDE VARIATIONS IN F8 PROMOTER REGION
usually increases FVIII:C levels twofold to sixfold [23,24]. Recently, a number of parameters including the molecular abnormality causing HA have been shown to influence DDAVP response [25,26]. In some cases, this treatment can be inadequate (partial response) or ineffective (no response) [28]. In this study, seven patients with a promoter mutation did not respond effectively to this treatment. There are no data for the remaining three patients. This is consistent with the study by Riccardi et al., demonstrating that the c.-257T>G mutation was associated with ineffective DDAVP therapy [15,27]. However, this resistance was also observed in 8 of 10 patients without F8 mutations [28]. DDAVP releases the VWF and FVIII contained in endothelial cells [29] but it is difficult to explain this lack of response, as the mechanisms causing DDAVP to mobilize FVIII are currently poorly understood. Our knowledge of promoter regions has greatly improved over the last 20 years. Numerous mutations identified in the promoters of other genes have been previously studied (factor IX and haemophilia B Leyden [30], protein C [31], haptoglobin [32] and explain the patients’ clinical phenotypes. However, molecular variations in the F8 promoter are rare and represent less than 0.1% of cases of HA [13,14]. These variations are not necessarily all causative and it is therefore necessary to prove their role in the onset of the disease. In this
References 1 White GC II, Rosendaal F, Aledort LM, Lusher JM, Rothschild C, Ingerslev J. Definitions in hemophilia. Recommendation of the scientific subcommittee on factor VIII and factor IX of the scientific and standardization committee of the International Society on Thrombosis and Haemostasis. Thromb Haemost 2001; 85: 560. 2 Vehar GA, Keyt B, Eaton D et al. Structure of human factor VIII. Nature 1984; 312: 337–42. 3 Gitschier J, Wood WI, Goralka TM et al. Characterization of the human factor VIII gene. Nature 1984; 312: 326–30. 4 Lakich D, Kazazian HH Jr, Antonarakis SE, Gitschier J. Inversions disrupting the factor VIII gene are a common cause of severe haemophilia A. Nat Genet 1993; 5: 236–41. 5 Antonarakis SE, Rossiter JP, Young M et al. Factor VIII gene inversions in severe hemophilia A: results of an international consortium study. Blood 1995; 86: 2206–12. 6 Bagnall RD, Waseem N, Green PM, Giannelli F. Recurrent inversion breaking intron 1 of the factor VIII gene is a frequent cause of severe hemophilia A. Blood 2002; 99: 168–74. 7 Riccardi F, Tagliaferri A, Manotti C, Pattacini C, Neri TM. Intron factor VIII gene inversion in a population of Italian hemophilia A patients. Blood 2002; 100: 3432.
© 2013 John Wiley & Sons Ltd
e155
study, only four of six nucleotide variations detected were considered as causative for HA. When molecular variation was detected in F8 promoter, we propose to use prediction software and to verify predictions by reporter gene analysis. If the mutation is causative, it will be probably associated with a lack of therapeutic response to DDAVP and this clinical implication should be considered by clinicians.
Acknowledgements Many thanks to Dr. Caron de Fromentel for the gift of the PLC/PRF/5 cell lines. This study would not have been possible without the help of Dorothee Pellechia, Dr Bertrand M, Dr. Monpoux F, Dr Roussel Robert, Dr Pierre Louis, Dr Lienhart A and Dr Meunier S, Dr Peynet and Dr Durand B. The authors thank Dr Enjolras N and Dr Plantier JL for their excellent scientific assistance.
Author contributions C. Nougier, O. Roualdes, M. Fretigny and C. Vinciguerra performed the research, C. Nougier and O. Roualdes, C. Costa, R. d’Oiron analysed the data and wrote the paper. C. Negrier, C. Vinciguerra, C. Nougier designed the research study.
Disclosures The authors stated that they have no interests that might be perceived as posing a conflict or bias.
8 Goodeve AC, Peake IR. The molecular basis of hemophilia A: genotype–phenotype relationships and inhibitor development. Semin Thromb Hemost 2003; 29: 23–30. 9 Ogata K, Selvaraj SR, Miao HZ, Pipe SW. Most factor VIII B domain missense mutations are unlikely to be causative mutations for severe hemophilia A: implications for genotyping. J Thromb Haemost 2011; 9: 1183–90. 10 McGlynn LK, Mueller CR, Begbie M, Notley CR, Lillicrap D. Role of the liverenriched transcription factor hepatocyte nuclear factor 1 in transcriptional regulation of the factor VIII gene. Mol Cell Biol 1996; 16: 1936–45. 11 Figueiredo MS, Brownlee GG. cis-acting elements and transcription factors involved in the promoter activity of the human factor VIII gene. J Biol Chem 1995; 270: 11828–38. 12 Bogdanova N, Markoff A, Eisert R et al. Spectrum of molecular defects and mutation detection rate in patients with mild and moderate hemophilia A. Hum Mutat 2007; 28: 54–60. 13 Dai L, Cutler JA, Savidge GF, Mitchell MJ. Characterization of a causative mutation of hemophilia A identified in the promoter region of the factor VIII gene (F8). J Thromb Haemost 2008; 6: 193–5. 14 Zimmermann MA, Meier D, Oldenburg J, Muller CR, Rost S. Identification and characterization of mutations in the promoter
15
16
17
18
19
20
21
region of the factor VIII gene. J Thromb Haemost 2012; 10: 314–7. Riccardi F, Rivolta GF, Franchini M, Pattacini C, Neri TM, Tagliaferri A. Characterization of a novel mutation in the F8 promoter region associated with mild hemophilia A and resistance to DDAVP therapy. J Thromb Haemost 2009; 7: 1234–5. Bagnall RD, Giannelli F, Green PM. Int22 h-related inversions causing hemophilia A: a novel insight into their origin and a new more discriminant PCR test for their detection. J Thromb Haemost 2006; 4: 591–8. Chekmenev DS, Haid C, Kel AE. P-Match: transcription factor binding site search by combining patterns and weight matrices. Nucleic Acids Res 2005; 33: W432–7. Wion KL, Kelly D, Summerfield JA, Tuddenham EG, Lawn RM. Distribution of factor VIII mRNA and antigen in human liver and other tissues. Nature 1985; 317: 726–9. Graw J, Brackmann HH, Oldenburg J, Schneppenheim R, Spannagl M, Schwaab R. Haemophilia A: from mutation analysis to new therapies. Nat Rev Genet 2005; 6: 488–501. Shi Y, Lee JS, Galvin KM. Everything you have ever wanted to know about Yin Yang 1. Biochim Biophys Acta 1997; 1332: F49– 66. Yokoyama NN, Pate KT, Sprowl S, Waterman ML. A role for YY1 in repression of
Haemophilia (2014), 20, e149--e156
e156
22
23
24
25
26
27
C. NOUGIER et al.
dominant negative LEF-1 expression in colon cancer. Nucleic Acids Res 2010; 38: 6375–88. Li JR, Li JG, Deng GH et al. A common promoter variant of TBX21 is associated with allele specific binding to Ying-Yang 1 and reduced gene expression. Scand J Immunol 2011; 73: 449–58. Mannucci PM. Desmopressin (DDAVP) for treatment of disorders of hemostasis. Prog Hemost Thromb 1986; 8: 19–45. Castaman G. Desmopressin for the treatment of haemophilia. Haemophilia 2008; 14(Suppl. 1): 15–20. Mannucci PM. Desmopressin (DDAVP) in the treatment of bleeding disorders: the first twenty years. Haemophilia 2000; 6(Suppl. 1): 60–7. Seary ME, Feldman D, Carcao MD. DDAVP responsiveness in children with mild or moderate haemophilia A correlates with age, endogenous FVIII:C level and with haemophilic genotype. Haemophilia 2012; 18: 50–5. Castaman G, Mancuso ME, Giacomelli SH et al. Molecular and phenotypic determi-
Haemophilia (2014), 20, e149--e156
28
29
30
31
nants of the response to desmopressin in adult patients with mild hemophilia A. J Thromb Haemost 2009; 7: 1824–31. Tagliaferri A, Di Perna C, Riccardi F, Pattacini C, Rivolta GF, Franchini M. The natural history of mild haemophilia: a 30year single centre experience. Haemophilia 2012; 18: 166–74. Xu L, Nichols TC, Sarkar R, McCorquodale S, Bellinger DA, Ponder KP. Absence of a desmopressin response after therapeutic expression of factor VIII in hemophilia A dogs with liver-directed neonatal gene therapy. Proc Natl Acad Sci U S A 2005; 102: 6080–5. Hirosawa S, Fahner JB, Salier JP, Wu CT, Lovrien EW, Kurachi K. Structural and functional basis of the developmental regulation of human coagulation factor IX gene: factor IX Leyden. Proc Natl Acad Sci U S A 1990; 87: 4421–5. Berg LP, Scopes DA, Alhaq A, Kakkar VV, Cooper DN. Disruption of a binding site for hepatocyte nuclear factor 1 in the protein C gene promoter is associated with hereditary thrombophilia. Hum Mol Genet 1994; 3: 2147–52.
32 Grant DJ, Maeda N. A base substitution in the promoter associated with the human haptoglobin 2-1 modified phenotype decreases transcriptional activity and responsiveness to interleukin-6 in human hepatoma cells. Am J Hum Genet 1993; 52: 974–80. 33 Margaglione M, Castaman G, Morfini M et al. The Italian AICE-Genetics hemophilia A database: results and correlation with clinical phenotype. Haematologica 2008; 93: 722–8. 34 Green P, Bagnall R, Waseem N, Giannelli F. Haemophilia A mutations in the UK: results of screening one-third of the population. Br J Haematol 2008; 143: 115–28. 35 Lombardi AM, Zanon E, Sartori MT, Cabrio L, Vettore S, Girolami A. Molecular genetics of bleeding disorders. Haemophilia 2004; 10(Suppl. 3): 73–78. 36 Sanna V, Zarrilli F, Nardiello P et al. Mutational spectrum of F8 gene and prothrombotic gene variants in haemophilia A patients from Southern Italy. Haemophilia 2008; 14: 796–803.
© 2013 John Wiley & Sons Ltd
